METHOD FOR GENERATION OF SEQUENCE SAMPLED MAPS OF COMPLEX GENOMES

Abstract: The present invention relates to a rapid and powerful sequence "sequence sampled mapping" method for sequencing complex genomes. The invention method is applicable to genomic DNA, preferably mammalian chromosomes, and in a preferred embodiment, employs a "bottom-up" mapping strategy, which allows for the simultaneous analysis of multiple cosmid clones for the detection of overlaps. The sequence sample mapping method is useful first, for the completion of high density sequence-based maps, and ultimately, for the complete sequencing of genomic DNA directly from cosmid clones.

Description: METHOD FOR GENERATION OF SEQUENCE SAMPLED MAPS OF COMPLEX GENOMES The present invention relates to recombinant DNA technology. More particularly, the invention concerns a process for rapidly generating a physical sequence map of large complex genomes, including human chromosomes. The sequence mapping process ("sequence sampled mapping") depends on the use of cosmid vectors containing endogenous bacteriophage promoters to allow for the sequencing of end-specific nucleotides of each member of a contiguous library of cosmid clones. Background of the Invention The complete analysis of large complex genomes, such as genomes of higher eukaryotes, including human, requires the extensive isolation, purification and analysis of large fragments of DNA by cloning, generally in E. coli . In the past, the lambda bacteriophage cloning system has been used most frequently to generate genomic libraries. The lambda bacteriophage vectors usually accommodate inserts up to about 20 kb. Presently the primary system used to clone and manipulate large DNA fragments is that of cosmid vectors. Cosmid vectors allow the packaging of DNA fragments of up to about 45 kb in piasmids containing bacteriophage cos sites for in vitro packaging. The analysis of complex genomes involves the application of both "top-down" and "bottom-up" mapping strategies. The "top-down" strategy depends on the separation on pulsed field gels of large DNA fragments generated using rare restriction endonucleases for physical linkage of DNA markers and the construction of long-range maps [Schwartz et al., Cell 3_7:67 (1984); Southern et al., Nucleic Acids Res. .15:5925 (1987); Burke et al., Science 236:806 (1987)]. The "bottom-up" strategy depends on identifying overlapping sequences in a large number of randomly selected bacteriophage or cosmid clones by unique restriction enzyme "fingerprinting" and their assembly into overlapping sets of clones. "Top down" mapping is inherently more rapid and less labor intensive, but does not generate sets of DNA clones for further structural or biological analysis. "Bottom-up" mapping generates the required sets of overlapping clones but application of current strategies and pattern matching algorithms to mammalian genomes will require the analysis of thousands to tens of thousands of individual clones for the generation of complete maps. Clone-based physical maps have been extremely useful as the framework for many types of structural and biological studies and have been constructed for several model organisms including E. coli , C. eleganε , D. melanogaster and S. cerevesiae (Kohara et al., 1989, Cell. 50:495-508; Oliver et al. , 1992, Nature. 357:38-46; Sulston et al., 1992, Nature. 356:37-41; Merriam et al., 1991, Science. 254:221-225). In the past few years, a variety of techniques have been utilized for the construction of ordered clone maps including cosmid and phage contig-building (Olson et al., 1986, Proc. Natl. Acad. Sci.. USA, 83:7826-7830; Coulson et al., 1986, Proc. Natl. Acad. Sci.. USA. 83:7821-7825), analysis of arrayed libraries (Evans and Lewis, 1989, Proc. Natl. Acad. Sci.. USA. 86:5030-5034), linking libraries and pulsed-field gel analysis (Poustka and Lehrach, 1986, Trends Genetics. 2:174-179; Hermanson et al. , 1992, Genomics. 13:134-143) and the assembly of YAC clone contigs (Bellanne-Chantelot et al., 1992, Cell. 70:1059-1068; Foote et al. , 1992, Science 258:60-66). Another approach for the assembly of clone maps is the use of sequence tagged sites (STSs; Olson et al, 1989, Science. 245:1434-1435): mapped DNA sequence fragments that can be detected by amplification of specific products using the polymerase chain reaction (PCR; Saiki et al., 1988, Science. 239:487-491). STS content mapping, the analysis of STS markers present in large contiguous inserts in yeast artificial chromosomes, has proven to be an efficient method for assembling clone maps of 100 to 300 kb average resolution and has been successful for the assembly of low resolution maps of human chromosomes Y and 21 (Foote et al, 1992, supra; Chu akov et al., 1992, Nature, 359:380-387). The analysis of STS content in large DNA fragments carried in somatic cell (Delattre et al., 1991, Genomics, 9:721-727) or radiation-reduced cell hybrid lines (Cox et al., 1990, Science. 250, 245-250) also provides a powerful mapping technique. Large numbers of mapped STS markers have been isolated for several human chromosomes (Tanigami et al., 1992, Am J Hum Genet. 50:56-64; Hori et al., 1992, Genomics. 13:129-133; Heding et al., 1992, Genomics, 13:89-94) but do not necessarily provide the needed resources for large scale chromosome mapping. In many cases, the value of these reagents is limited because the probes are poorly characterized, not generally available to the scientific community or can not be used for routine screening under a set of standardized conditions. Thus, methods of producing DNA markers suitable as reagents for large scale chromosome mapping are desired. In addition, a major challenge of the human genome project is development of new approaches for physical analysis and sequence determination. Major progress has been made with the sequencing of large regions of DNA for significant portions of the E. coli genome, chromosome III of S . cereviεiae , several cosmid sized pieces of DNA from C. elegans , human, and a 100 kb T cell receptor region from mouse. See, e.g., Daniels et al., Science. 257:771-778 (1992); Martin-Gallardo et al., Nat. Genet. 1:34-39 (1992); Oliver et al.. Nature. 357:38-46 (1992); Sulston et al. , Nature. 356:37-41 (1992); and Wilson et al., Genomics. 13:1198-1208 (1992). The precise determination of each base for these sequences, however, has been a labor intensive and costly undertaking. In addition, the construction of high resolution physical maps and the acquisition of sequence have previously been considered separate efforts. Thus, new methods are desired, such as combining the steps of physical mapping and sequencing, to sequence 25%-100% of a particular portion of genomic DNA from megabase sized regions to whole genomes or chromosomes more economically than previous efforts and with reasonable accuracy. Summary of the Invention The present invention relates to a rapid and powerful sequence mapping method, called "sequence sampled mapping", for sequencing complex genome, said method comprising sequencing the end-specific nucleotides of each member of a library of cosmid clones, and assembling a sequence sampled map by correlating the end- specific sequence information with the relative spatial relationship between the cosmids. The invention method is applicable to genomic DNA, preferably mammalian chromosomes, and in a preferred embodiment, employs a "bottom-up" mapping strategy, which allows for the simultaneous analysis of multiple cosmid clones for the detection of overlaps. The sequence sampled mapping method permits sequence overlaps to be determined by map positions, reducing the reliance on determining regions of unique shared sequence. In a particular embodiment of the invention, the method comprises, in any order, grouping of cosmid clones by chromosome or YAC hybridization, construction of high density cosmid contigs by restriction based fingerprinting, and direct and automated DNA sequencing from cosmid clones. The sequence sampled mapping method is useful for the completion of high density sequence-based maps, and ultimately, for the complete sequencing of genomic DNA directly from cosmid clones. In addition, the resulting sequence information allows the detection of many genes by sequence analysis with computer programs such as FASTA, BLAST, GRAIL and others under development; allows the development of sequence tag sites (STSs) and polymorphic repeats at an actual physical spacing of a few kilobases (see, e.g., Olson et al., 1989, Science. 245:1434-1435); and allows direct PCR amplification of any part of the genome, independent of clone libraries. The invention method is also amenable to automation using the particular characteristics of the sCOS vector and cloning system. The resulting sequence sampled map is also useful, employing on-line parallel processing microcomputers which use existing software programs that have been adapted for parallel processing, for the computer analysis of genomic DNA. Brief Description of Drawings Figure 1 shows the vector sCOS-1 designed for cosmid multiplex analysis. The vector contains bacteriophage T3 and T7 promoters flanking a unique BamHI cloning site, NotI sites for expedited restriction mapping and excision of the insert DNA, duplicated cos sites for high efficiency microcloning, a dominant selection for transfection into mammalian cells. Amp and Kn resistance genes, and ColEl origin of replication. Figure 2 illustrates the construction of cosmid vector sCOS-1. Relevant restriction sites in the precursor molecules are shown. Clal-Sall and Clal-Xhol fragments were excised from pWE15 and pDVcosl43 and purified on agarose gels. The indicated fragments were joined using T4 DNA ligase and co-ligation of the Xhol and Sail sites resulted in the loss of both sites in the resulting piasmids. Figure 3 depicts the DNA sequences of the cloning site, bacteriophage promoters and flanking restriction sites in sCOS vectors. Restriction sites and T3 and T7 promoter sequences added using synthetic oligonucleotides are shown. Sfil , NotI , EcoKL and SacII restriction sites are indicated by thin lines. The direction of transcription using T3 or T7 polymerase is indicated by the arrows and the thick lines delineate the critical nucleotides for promoter activity. The BamHI site is the cloning site into which Mbol-digested genomic DNA is inserted. All linkers were inserted by "linker-tailing" into the sites formed by digestion of sCOS-1 with EcoRI. Figure 4 illustrates a strategy useful for analysis of physical linkage using groups of cosmids. Figure 4A illustrates that cosmids prepared in vector sCOS-1 or one of its derivatives can be used to synthesize end-specific sequences (e.g., probes for the detection of overlaps) . Figure 4B illustrates the inoculation of cosmid clones on the surface of a nitrocellulose or nylon filter from 96-well archive plates stored at -70°C. Each clone on the "grid" is assigned a unique identifying Y and X axis coordinate. Individual clones in the collection contain the innate capacity of generating probes specific for the extreme ends of the genomic DNA insert and detecting overlapping clones on the filter. The arrows show the locations of potential overlapping clones detected by hybridization of probes generated from the clone at coordinates Y = 2, X = 7. Figures 4C and 4D illustrate the analysis of multiple clones simultaneously. Cosmids are pooled according to the rows and columns or the matrix, DNA prepared and a mixed RNA probe synthesized. When hybridized to the matrix filter, the probe detects a pattern of spots consisting of all of the template clones and the collection of clones overlapping with one end of each of the template clones. A similar, procedure is carried out using cosmids pooled according to columns of the matrix. When the two data sets are compared, hybridizing clones identified by both of the mixed probes may be overlapping with the template clone common to both sets: that clone located at the intersection of the row and column. This procedure may then be repeated using other combinations of pooled probes and either T7 or T3 polymerase. The arrows denote the location of a clone which overlaps with the "T7 end" of the clone at coordinates Y = 2, X = 4. Figure 5 shows predicted contigs from human chromosome llq and restriction enzyme digestion analysis. Figure 5A presents the predicted linkage and orientation of a representative cosmid contig generated by multiplex analysis of the chromosome llq cosmid set and data analysis using the computer program "Contig- aker". The computer output indicates the coordinates of linked clones (X,Y) and the arrows denote the orientation of the linkage. Figure 5B presents a restriction map and the location of probes used to establish unequivocal overlap of the cosmids. A restriction map of the overlapping clones detected in Figure 5A was determined by the analysis of partial EcoRI digestion products hybridized with P-labeled T3 or T7 promoter-specific oligonucleotides. Overlapping areas not confirmed by restriction map analysis were confirmed by hybridization analysis using end-specific RNA probes generated from individual cosmid clones. Cosmid clones cl4,23 and cl9,27 are identical. D indicates bacteriophage T3 promoter, ■ indicates bacteriophage T7 promoter. Figure 6 presents physical and sequence maps of the (A) / ff-giardin and (B) a second random genomic region with arrows at the ends of cosmids corresponding to their t7 ends. Smaller hash marks represent locally regionalized, but not fully ordered restriction fragments. Regions of sequence which match the genes annotated on the figure are shown with a greyed-in box. Figures 7A-F present alignment of various regions of sequence homology found by BLAST searches of the protein sequence databanks with cosmid end sequences. Figure 8 presents a histogram showing the distances between ordered cosmid ends and their frequency of occurrence in the two contigs determined as described herein. Figure 9 presents a plot of the number of sequenced cosmid ends versus the total amount of Giardia lamblia genome sequenced. Calculations are based on the equations of Lander and Waterman [Genomics, 2:231-239 (1988)] assuming an overlap detection of 50 bases. Figure 10 presents regional mapping of a chromosome 11 STS using a panel of somatic cell hybrids. This analysis shows the regional mapping of an STS to bin 2, FLpter 0.05-0.24, by PCR analysis and is typical of the bulk of STS primer results derived under these conditions. The hybrid mapping panel breakpoints are shown relative to human chromosome 11. Figure 11 presents protein sequence alignments of putative genes detected from analysis of DNA sequences. DNA sequence determined from cosmid clones were translated into six reading frames and used to search GenPept, PIR or Swiss-Prot protein sequence databases using BLASTX. The clone name from which the sequence was derived is shown next to its translated sequence. The flanking numbers indicate the matching position in the nucleotide sequence of the cosmid or the protein sequence of the database entry. The one amino acid code translation is shown with X=any amino acid (generally caused by the inability to determine a base) and = a stop codon. The X in cSRL-7d2 has a one in four chance of being a stop codon. Detailed Description of the Invention In accordance with the present invention, there is provided a method for sequencing complex genomes. The invention method comprises: (1) sequencing the end-specific nucleotides of each member of a library of cosmid clones, wherein said cosmid clones are prepared by inserting genomic DNA fragments into cosmid vectors, wherein the cosmid vectors include sequences of nucleotides that flank at least one end of the inserted DNA, and that serve as transcription initiation sites for the synthesis of a nucleic acid specific to the ends of the inserted DNA, and (2) assembling a sequence sampled map by correlating the end-specific sequence information with the relative spatial relationship between the cosmids. In a preferred embodiment, the invention sequence sampled mapping method provides for the sequencing of the entire genome of any organism for which genomic DNA is available, preferably mammalian genomic DNA, more preferably human genomic DNA. As used herein, the phrase "end-specific nucleotides of each member of a library of cosmid clones" refers to the nucleotide sequences at the extreme 5' and 3' ends of a given genomic DNA insert. Typically, the amount of nucleotides sequenced from each end-specific nucleotide sequence will be at least 100, preferably 250, more preferably 350, yet more preferably 550, with at least 1000 nucleotides being especially preferred. The amount of sequenced nucleotides required for the practice of the invention method varies as a function of the depth of cosmids to be sequenced. The phrase "depth of cosmids", and grammatical variations thereof, refers to the number of overlapping cosmids that contain, in common, a specified region (i.e., 1 nucleotide) of genomic DNA to be mapped and sequenced. For example, a 2OX (20-fold) depth of cosmids covering a specified region of genomic DNA refers to 20 cosmids that, on average, contain at least one nucleotide of genomic DNA in common. The depth of cosmids is chosen so as to maximize the number of unique genomic DNA insert ends and to provide a desired average spacing between the respective 5' or 3' ends of two consecutive contiguous cosmid clones. For example, if on average, each cosmid contains approximately 40 kb of genomic DNA insert, then a cosmid depth of 2OX would produce, on average, a spacing of 1000 nucleotide base pairs between the respective 5* or 3' ends of each consecutive genomic DNA insert. Thus, sequencing approximately 500 base pairs of the 5• and 3' ends of all given cosmids will provide genomic DNA sequence data for approximately 50% of the respective genomic DNA sample. The depth of cosmids can be varied by methods well-known in the art. One method is to select any one or combination of restriction enzymes that recognize a specific genomic DNA sequence, preferably a 4-bp sequence ("4-bp-recognizing") . Next the restriction enzyme(s) are employed to either partially or completely digest a given genomic DNA sample so as to produce genomic DNA insert fragments approximately 40-45 kb in length that are unique with respect to other genomic DNA insert fragments by as little as 100 base pairs to as great as 5 - 10 kb. Restriction enzymes that recognize 4 bp sequences suitable for use herein include, for example: Sau3A, AccII, Alul, BSP50, FnuDII, Haelll, Hhal, the isoschizomers thereof, and the like. A twenty-fold library generated by partial digestions of genomic DNA with several different restrictions enzymes would greatly increase the number of potential cloning sites and reduce the number of cloned ends which are exactly the same. For example, multiple 5-10 fold deep libraries, from the same genomic DNA source, can each be generated with a unique four-bp- recognizing restriction enzyme. This would require straightforward modifications to the COS vectors described hereinater, e.g., adding appropriate polylinker sites. The uniquely restricted libraries can then be combined to arrive at libraries with a substantially high level of cosmid depth (e.g., at least about 20-fold deep, preferably at least about 40-fold deep, and more preferably at least about 50-fold deep) . Thus, a 21-fold library may be constructed from three sub-libraries of 7- fold cosmid depth, whereby each sub-library is produced with a different four-bp-recognizing restriction enzyme. One of skill in the art will recognize that by varying any one or both of the above-described parameters, sequencing of varying percentages of a given genomic sample becomes feasible, such as at least 25%, preferably 50%, more preferably 75%, with 100% (i.e., the entire genome) being especially preferred (see, e.g., Figure 9) . For example, in the above-example, increasing the depth of cosmids from 20X to 40X and sequencing 500 bp of each end of genomic DNA inserts provides the sequence for 100% of the 40 kb genomic DNA sample. Alternatively, in the above-example, increasing the amount of nucleotides sequenced from 500 to 1000 bp also provides the sequence for 100% of the 40 kb genomic DNA sample. Stated another way, increasing the average lengths of sequences determined, to about one kilobase, would result in nearly complete one-pass sequencing of a genome or chromosome at a fraction of the cost. Either prior to, concurrently, or following the construction of contigs or determining the relative spatial relationship between the cosmid clones described above, the sequencing of end-specific nucleotides step of the present invention, is conducted to provide the sequence information that will be assembled into the "sequence sampled map". The sequencing step may be carried out either manually or using an automated DNA Sequencer employing well-known methods, such as, for example, specific or degenerate primer extension, transposon primer insertion, ordered deletion, random shot gun sequencing, sequencing by hybridization, and the like. In a preferred embodiment, the 5' and 3' ends of each cosmid clone within a cosmid library is subjected to "one pass" (i.e., sequencing only once) automated DNA sequencing as described in Examples 3 and 4. Automated DNA sequencing devices are well-known and widely available to those of skill in the art, such as, for example, the sequencing devices available from Applied Biosystems (e.g., ABI 373A Sequencer combined with a Catalyst 800 robot, Foster City, CA) , Pharmacia (Piscataway, NJ) , Millipore (Milford, MA), and the like. It is recognized that automated DNA sequencing technology is currently progressing extremely rapidly. Thus, automated sequencing methods and devices that will allow sequencing of DNA fragments greater than 500 nucleotides (i.e., 1 to 5 kb) are also contemplated in the methods described herein (see, e.g., Ansorge et al., 1992, Electrophoresis. 13:616-619). Subsequently, when correction and verification of sequence information is desired for a particular region, an independent sequencing methodology may be employed, e.g., sequencing by hybridization, and the like. Raw sequence information obtained from automated sequencing, or sequence sampled mapped sequence, can be analyzed immediately using on-line parallel processing microcomputers that employ existing software programs adapted for parallel processing. Sequence analysis software programs contemplated for use herein include, for example: GRAIL, which locates protein-coding regions in genomic DNA sequences (see, e.g., Uberbacher et al., PNAS, USA, 88:11261-11265, 1991) ; BLAST-n and BLAST-x, which compares sequence similarity between nucleotides and amino acid sequences, respectively (see, e.g., Altschul et al., J. Mol. Biol., 215:403-410, 1990); FASTA, which identifies sequence repeats (see, e.g., Pearson et al., PNAS, USA, 85:2444- 2448, 1988). Raw sequence information may also be used advantagously to generate PCR primers useful in PCR assays for polymorphic repeats around specific sequences of interest, e.g., around "CA" nucleotide runs, other simple sequences, and the like. In another aspect of the invention, prior to the completion of a complete physical map, raw sequence information can be used to generate "sequence-tagged sites" (STSs) as described in Example 4. The STSs can be used, e.g., for producing an ordered set of YACs, for the analysis of sites of chromosomal pathology (e.g., translocations, polymorphic repeats, and inversions) , and the like. The production of STSs allows access to mapping markers based upon PCR amplification of known genomic sequence. Briefly, as described above, DNA sequences are determined by sequencing directly from cosmid templates using primers complementary to the promoters (e.g., T3 and T7) present in the cloning vector. Oligonucleotide PCR primers are predicted by computer from a suitable amount of randomly selected cosmid-end-derived sequences, and are tested using a battery of genomic DNA templates, preferably corresponding to a specific chromosome. Cosmids are then regionally localized to the respective chromosome using fluorescence in situ hybridization and/or by the analysis of a somatic cell hybrid panel. Additional STSs corresponding to known genes and genetic markers on the respective chromosome may also be produced under the same series of standardized conditions. As used herein, a "suitable amount" of STSs produced can be varied by one of skill in the art to provide a desired coverage (preferably uniform) of a respective chromosome. For example, it is well within the skill in the art to select an amount of STSs that provides an average spacing between conscecutive STSs within the range of about 1 kb up to about 500 kb, and also provides sufficient density for STS content mapping using YAC clones or contigs (see, e.g., Bellanne-Chaltelot et al., 1992, Cell. 70:1059-1068). Thus, e.g., assuming a chromosomal size of 126 mb (e.g., based upon chromosome 11 comprising 4.2% of the human genome) , a collection of 370 STSs will have an average spacing of one STS per 340 kb and would provide sufficient density for STS content mapping using YAC clones. In a preferred embodiment, the quantity of STSs produced by the methods described herein provides an average spacing between conscecutive STSs within the range of about 1 kb up to about 5 kb. As used herein, the phrase "assembling a sequence sampled map" refers to the step of ordering the nucleotide sequences obtained from the sequencing step into the order in which they naturally occur in the source genome. This is accomplished by correlating the end-specific sequence information obtained in step (1) above with the relative spatial relationship between the cosmids. Once the sequence sampled map has been determined, a minimum tiling path of cosmids (just enough to cover the region once) can be used as sequencing templates. Each sampled sequence can then be extended in both directions to triple the effective sequence reads. The availability of inexpensive oligonucleotide primers will make this sequence walking an attractive option for finishing the sequence. One attractive approach which may make primer walking affordable is the use of contiguous hexamer oligonucleotides to specifically prime sequencing reactions (see, e.g., Kieleczawa et. al., 1992, Science. 258:1787-91). The term "relative spatial relationship between the cosmids" refers to the physical mapping of the genomic DNA inserts into the contiguous order and/or the respective orientations (e.g., 5'-3' or 3'-5') in which they naturally occur in the source genome. The relative spatial relationship may be determined either prior to, concurrently, or after the step of "sequencing the end-specific sequences of each member of a library of cosmid clones." In a preferred embodiment, the relative spatial relationship between the cosmids is determined concurrent (i.e., in parallel) with the sequencing of step (1) above. The relative spatial relationship between the cosmids (i.e., physical map) can be determined by methods well-known in the art, such as fingerprinting by restriction enzyme mapping ("restriction-fragment-length mapping") employing several different restriction enzymes [see, e.g., Olson et al., Proc. Natl. Acad. Sci. USA i$3.:7826 (1986); Coulson et al., Proc. Natl. Acad. Sci. USA 83.:7821 (1986); Kohara et al., Cell 50:495 (1987); and the like] ; the "cosmid multiplex analysis" method as described in U.S. Patent No. 5,219,726, incorporated herein by reference in its entirety; and the like. Restriction fragment length mapping employs full and/or partial restriction enzyme digests. The partial digestion provides the order of restriction within a cloned genomic insert, and the full digestion provides the exact sizes of the restriction fragments. Partial digestion products are detected with non-insert probes specific to either side of the cosmid vector, which orients the sequence relative to the genomic map. The partial digestion restriction maps are reconciled with data from the full restriction digest to obtain a spatially correct physical map. The restriction enzyme maps of each cosmid are compiled into an overall map of the genomic region of interest, which could be derived from a YAC (Yeast Artificial Chromosome) , BAC (Bacterial Artificial Chromosome) , PAC (Pi Artificial Chromosome) , MAC (Mammalian Artificial Chromosome) , a whole chromosome, or a small whole genome. In another embodiment of the present invention, the relative spatial relationship between the cosmids is determined by the "cosmid multiplex analysis" method as described in Example 2 and U.S. Patent No. 5,219,726. The cosmid multiplex analysis method depends on the use of cosmid vectors allowing for the synthesis of corresponding RNA sequences (probes) or DNA sequences specific to the extreme ends of the DNA fragments contained within the cosmid, directly from the DNA inserts of the cosmid. Briefly, cosmid libraries are constructed using vectors containing at least one bacteriophage promoter adjacent to the genomic DNA insert, positioned operatively for the transcription thereof. Preferably, the cosmid vectors contain two bacteriophage promoters flanking the DNA fragment ligated into the insertion site. Synthesis of an end-specific RNA probe from any clone in the collection allows the overlapping clones to be easily detected by hybridization. Because this strategy does not depend on pattern recognition for detecting overlaps, analysis may be carried out simultaneously on cosmid clones grouped together. The "cosmid multiplex analysis" method is suitable for the unambiguous detection of overlapping regions as small as several hundred nucleotides in contiguous cosmids. Accordingly, the number of clones needed for map closure can be reduced by up to three-fold. Finally, this strategy represents essentially simultaneous cosmid "walking" and thus is basically non-random, allowing the investigator the freedom to pause and investigate some interesting biology rather than requiring completion of the map before it becomes useful. It has been found that significant improvements in the speed and efficiency of "bottom-up" genomic mapping can be achieved, by 1) isolating restricted regions of large mammalian genomes in a "sublibrary" preorganized on a solid matrix, 2) using hybridization of end-specific probes for detection of overlapping clones in the collection, and 3) analyzing multiple clones simultaneously for the detection of all. overlaps in the collection. In accordance with the present invention, direct sequencing can then be carried out on the fragment ends of the individual cosmids which make up the resulting contig. This will generate in the range of about 350-550 base pairs of sequence information, separated by gaps of about 1-5 kb, depending on the chosen depth of cosmids employed. Thus, for a cosmid depth of 10-2OX, in the range of about 20-50% of the complete chromosome sequence can be obtained very rapidly and at relatively low cost. Thus, an alternate embodiment of the present invention provides a method for sequencing complex genomes, said method comprising: (1) preparing a genomic library of cosmid clones by inserting DNA fragments from said genome into cosmid vectors, wherein the cosmid vectors include sequences of nucleotides that flank at least one end of the inserted DNA, and that serve as transcription initiation sites for the synthesis of end-specific probes, (2) arranging the cosmid clones, whereby each clone may be identified and replicas of said arrangement may be reproduced, (3) pooling portions of said cosmid clones and synthesizing pools of mixed end-specific probes from the DNA inserts that have been prepared from said pooled clones, wherein each pool contains fewer than all of the cosmid clones in the library, but all of the cosmid clones in the library are included in at least one pool, (4) hybridizing each pool of probes to a replica of said arranged cosmid clones and identifying the cosmid clones in each replica that hybridize to the probes, wherein said identified clones include the pooled cosmid clones and cosmid clones that contain DNA inserts that overlap with the DNA inserts in the pooled clones, (5) identifying the cosmid clones from among those identified in step (4) that hybridize to two or more pools of probes, thereby identifying groups of cosmid clones that include overlapping DNA, (6) assembling contigs from said groups, and (7) sequencing the end-specific nucleotides of each overlapping member of said cosmid clones. In a preferred embodiment, the cross-hybridizing clones are identified by pairwise comparison of data sets obtained from two groups of cosmid clones containing at least one common clone. The cosmid clones are preferably pooled according to the rows and columns of a two-dimensional matrix. Preferably, the cosmid vectors used in the above processes comprise two oppositely oriented promoters, each of which is specific for a bacteriophage RNA polymerase, positioned on two sides of the cloning site. Most preferably, the vectors contain T3 and T7 endogenous bacteriophage promoters flanking the cloned genomic DNA. Vectors containing at least two cos sites are particularly preferred, since they allow the use of DNA fragments without the need for prior size separation. From the list of linked clones produced by this technique, contigs can be assembled either manually or through computer analysis of the data, then the fragment ends of the DNA inserts in the individual cosmids can be subjected to automated DNA sequencing. This will typically generate ordered sequence fragments about 350-550 base pairs in length, separated by gaps in the range of about 1-5 kb, depending on the chosen cosmid depth. As used herein, the term "genomic library" refers to a mixture of clones constructed by inserting fragments of genomic DNA into a suitable vector. The term "library" implies the existence of large numbers of different recombinants out of which only a few are of immediate interest to the investigator. Genomic DNA can be the entire genome, a single chromosome, or a portion of a chromosome, such as the 300-400 kb portions of chromosomal DNA typically contained within YACs, of a given organism. The terms "cosmid" and "cosmid vector" and grammatical variations thereof, are used interchangeably herein and refer to plasmid vectors that contain a lambda bacteriophage cos (cohesive end) site. The lambda bacteriophage packaging system selects DNA molecules of about the size of the lambda genome (37-52 kb) . Accordingly, plasmid recombinant DNA having a minimum size of about 38 kb and a maximum size of about 52 kb (about 78% and about 105% of phage lambda, respectively) , can be packaged in vitro in the lambda phage coat. In addition to the cos site(s) cosmid vectors usually contain a marker gene allowing for selection in bacteria (antibiotic resistance gene) , and one or more unique restriction sites for cloning. Piasmids with a large variety of cloning sites and prokaryotic and eukaryotic selection markers can be converted to cosmids by insertion of the lambda cos region. The term "cosmid clone" refers to a cosmid vector that contains a genomic DNA insert. The term "plasmid" refers to circular, double-stranded DNA loops which in their vector form, are not bound to the chromosome. The term "nucleic acid" refers to a synthetic or naturally occurring DNA or RNA molecule. As used herein, the term "a promoter specific for a bacteriophage RNA polymerase" means a wild-type or non-wild-type promoter that can be used by the bacteriophage RNA polymerase for in vitro transcription of a DNA fragment. When a non-wild-type promoter is used for such in vitro transcription of a DNA fragment, transcription will occur at a rate which is at least 10% of the rate at which transcription would have occurred if a wild-type or native promoter had been used by the bacteriophage RNA polymerase to transcribe the DNA fragment in vitro. The term "cloning site" as used herein, means restriction endonuclease site on the DNA sequence of the cosmid vectors of the present invention where a DNA fragment can be inserted without deleting any of the original DNA. Reference to a promoter positioned "operatively for transcription of a DNA fragment", as used herein, means that the promoter will be positioned in such a way that any DNA sequences between the promoter's transcriptional start site and the DNA fragment will not prevent transcription of at least a portion of the DNA fragment by the promoter. The term "at least a portion" means that preferably at least 8 base pairs and more preferably at least about 30 bp of the DNA fragment will be transcribed. The terms "end-specific RNA sequences", "RNA probes", and grammatical variations thereof, are used to refer to hybridization probes obtained by transcription of corresponding DNA fragments. Clones are overlapping if they contain contiguous DNA in the same relationship as that in the genome. One method for detecting overlaps is to synthesize an RNA probe from one end of. a first clone. If this probe detectably hybridizes with an end of the second clone under standard hybridization conditions, the two clones are overlapping [Wahl et al., PNAS USA 84:2160 (1987)]. The term "contig" was introduced by Rodger Staden, Nucleic Acids Res. 8:3673 (1980) in connection with DNA sequence analysis, and refers to groups of clones with contiguous nucleotide sequences. The cosmid multiplex analysis method employs essentially the strategy illustrated in Figure 4 used for genomic mapping using cosmid vectors. In a first step, a genomic library which represents a limited portion of a genome is constructed in a cosmid vector allowing for the synthesis of RNA probes and DNA strands for sequencing directly from insert DNA using endogenous bacteriophage promoters. A convenient and powerful way of subdividing the human genome for the preparation of libraries is through chromosome purification by flow cytometry [Gray et al., Cold Spring Harbor Symp. LI 1986 p. 141] . Cosmid vectors suitable for constructing a genomic "sub-library" include the pWE vectors described by Wahl et al., in Proc. Natl. Acad. Sci. USA 84:2160 (1987), e.g. pWE2, pWE4, pWE8 pWElO, pWE15, and pWE16, preferably pWE15 and pWE16. The construction of these vectors is described in the Materials and Methods section of the cited article and in Evans et al., Methods in Enzymology 152:604 (1987). These vectors, in addition to replication and selection functions, such as plasmid origin of replication, bacterial genes specifying antibiotic resistance, and the bacteriophage lambda cohesive termini (cos sequences) , contain the transcription promoters from either bacteriophage SP6, T7 or T3 flanking a unique BamHI cloning site. In one embodiment, cosmid vectors containing a duplicated cos sequence are employed. These "sCOS" vectors have the following important characteristics: 1) the presence of two cos sites such that packaging could be carried out with high efficiency and without requiring size selection of the insert DNA; 2) the presence of T3 and T7 bacteriophage promoters for the synthesis of "walking" probes; 3) unique restriction sites for removing the insert and to aid in restriction mapping; 4) selectable genes for gene transfer in eukaryotic cells; and 5) a plasmid origin of replication giving a high yield of cosmid DNA for preparing templates. The construction of plasmid sCOS-1 is described in the Examples, and is illustrated in Figures 1 and 2. The design of this plasmid (and derivatives thereof) allows for rapid production of RNA probes specific for both ends of the inserted DNA sequences. In addition, this design allows for the automated sequencing of the 5' and 3• ends of the genomic DNA insert. Plasmid vector, sCOS-1, shown in Figure 1, is 6.7 kb in size and has a cloning capacity of 31 to 48 kb. As with pWE vectors, bacteriophage T3 and T7 promoters were oriented into the BamHI cloning site to allow direct synthesis of end-specific RNA probes for molecular "walking" and to allow sequencing the end-specific nucleotides of a given genomic DNA insert. Previous experience with pWE cosmids suggested that WotI restriction sites may not be ideal for excision or mapping of inserts in some regions of the genome where WotI sites might be clustered. Therefore, additional cosmid vectors with other rare restriction sites have been constructed, by substituting the cloning/polymerase sites of sCOS-1 with sequences containing WotI and SacII sites (sCOS-2) or Sfil sites (sCOS-4) . The asymmetric rare sites in sCOS-2 are useful for cloning ends of large NotI or SacII fragments for isolation of "linking" clones for long range mapping by pulsed field gel analysis [Buiting et al., Genomics 2:143 (1988)]. Also, vectors which lack WotI sites, such as sCOS-4, would potentially allow the selection of clones containing unique WotI junction fragments by hybridization with Wotl-specific oligonucleotides [Estivill et al., Nucleic Acids Res. 15:1415 (1987)]. The double cos site sCOS vectors make feasible the preparation of representative libraries from very small amounts of purified, partially digested DNA, and are, therefore, presently preferred for carrying out the method of the present invention. One of skill in the art can modify the sCOS vectors employed herein in a variety of ways to make them more suitable for restriction-fragment-length mapping using partial digestion (see, e.g., Kohara et al.. Cell 5J):495-508 (1987). For example, an "sCOS-derivative" vector useful in the invention methods described herein can include, intron encoded endonuclease sites which recognize approximately 20 base pairs, and a multiple cloning site (i.e., polylinker) which will ligate with partially-digested genomic DNA from most commercially available restriction enzymes that recognize four bases and generate staggered ends. For determining the relative spatial relationship between the cosmid clones, the individual clones of the genomic library are arranged on a nitrocellulose or nylon filter matrix and each clone is identified by unique coordinates. If the randomly chosen clones are arranged in a two-dimensional matrix, they are identified by unique X and Y coordinates. For convenience in handling, the pattern of the matrix is preferably based on the pattern and spacing of wells of a standard 96-well microtitre plate and the repetitive preparation of culture plates and hybridization filters may be carried out using equipment designed for working with this standard. Each individual cosmid clone in the collection possesses the innate mechanism of generating an RNA probe capable of detecting any overlapping or identical clones in the collection. If an RNA probe is generated using T3 or T7 polymerase, and overlapping clones are detected by hybridization of the probe to a replica of the filter grid, using cosmid clones arranged on a 36 x 36 matrix containing 1296 clones, all of the overlaps can be detected by carrying out 1296 T3 polymerase reactions, 1296 T7 reactions and subsequent hybridization reactions. However, as an alternative to the individual analysis of cosmid clones for the detection of overlaps, simultaneous analysis of multiple cosmid clones in groups can be conducted, as described in U.S. Patent No. 5,219,726. Accordingly, this preferred strategy allows the analysis of a collection of cosmid clones with far less effort than "fingerprinting" each of the clones selected individually. The linked clones detected by the above method can then be grouped into contigs, either manually or, preferably, using appropriate computer programs. To confirm the correctness of the groupings, some of the contigs can be subjected to detailed restriction enzyme analysis, and the degree of physical overlap along with a physical map can be determined. To complete a physical genomic map, the above-outlined procedure can be repeated with as many clones as necessary, and the gaps between the contigs can be filled in, e.g. by traditional chromosome walking. The method described hereinabove represents a special case of a more general sequence mapping strategy based on clone matrices of higher order, such as, for example, greater than or equal to 3 dimensions (as described in U.S. Patent No. 5,219,726). Table 1 Theoretical analysis of genomes of various sizes by cosmid multiplex analysis where clones are organized in matrices of various dimensions and clones analyzed using probes prepared from groups of cosmid clones. * Where a matrix of n dimensions containing d n cosmids is used for the arrangement of cosmid clones , pooled probes of d" "1 members are prepared which allow for the analysis of d n individual clones by multiplex analysis using n x d mixed probes . For example , using a three dimensional matrix of 10 x 10 members , pooled probes of 100 individual clones could be used to analyze 1000 individual cosmids with 30 analytical reactions . Given that the theoretical limit of detectable signal from pooled RNA probes is not reached, cosmids representing the entire human genome with four-fold redundancy can potentially be analyzed by the "multiplex" using a 5-dimensional matrix of n=13 in. only 65 hybridization reactions . The theoretical limitation of this strategy seems to be the number of individual clones which can be pooled and still give reproducible hybridization signal . Current protocols suggest that this limitation may be somewhat greater than 100 clones. Further details of the invention are illustrated by the following, non-limiting examples. Examples Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1982) ; Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed... Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1989); Davis et al., Basic Methods in Molecular Biology. Elsevier Science Publishing, Inc. , New York, USA (1986) ; or Methods in Enzvmology: Guide to Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987) . Cosmid vectors Genomic libraries were constructed in cosmid vector sCOS-1 (illustrated in Figure 1) . sCOS-1 was prepared from cosmid vectors pWE15 [see Evans et al., Methods in Enzymology 152:604 (1987)]; ATCC Accession No. 37503, American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 USA] and pDVcosl34 [a gift from J. Reese, in wide circulation among scientists] . pWE15 DNA was digested with Clal and Sail , and the 6kb Clal-Sall restriction fragment, lacking the cos sequence was purified. Cosmid pDVcosl34 was digested with Clal and X ol and a restriction fragment containing the duplicated cos region was purified on a low melting point agarose gel. The purified fragments were ligated using T4 DNA ligase and transformed into E. coli host strain DH5, which is a derivative of the strongly recA " strain DH1 (commercially available, e.g. from Bethesda Laboratories, Gaithersburg, MD, USA) . Alternatively, purified fragments can be transformed into such host cells as AG1 (Stratagene Cloning Systems, San Diego, CA) , a derivative of DH5 selected for high packaging efficiency, or into HB101 (commercially available, e.g. from Bethesda Laboratories, Gaithersburg, MD, USA) . Other pWE piasmids suitable for genomic mapping according to the invention are disclosed in Evans et al., Methods in Enzymology, Supra. Cosmid vector pWE16 has been deposited with the American Type Culture Collection, and has been accorded ATCC No. 37524. Cosmids sCOS-2 and sCOS-4 are derivatives of sCOS-1 where the cloning site has been altered to substitute other rare restriction sites for the WotI sites. Cosmid vector sCOS-2 was constructed by digesting sCOS-1 with EcoRI, and purifying the plasmid DNA away from the WotI-T3 promoter-BamHI-T7 promoter-Wotl linker sequence by ethanol precipitation. A 30-nucleotide double-stranded synthetic oligomer with EcoRI coadhesive ends, containing WotI-T3 promoter-Ba__ιHI-T7 promoter-SacII sequences was added by linker-tailing [Lathe et al., DNA :173 (1984)]. sCOS-4 was constructed using a similar procedure adding a double-stranded synthetic oligonucleotide containing EcoRI coadhesive ends and a S_fil-T3 promoter-Ba_7iHI-T7 promoter-Sfil sequence. EXAMPLE 1 Construction of cosmid libraries in sCOS vectors High molecular weight genomic DNA for cosmid cloning was prepared by proteinase k digestion and gentle phenol extraction followed by dialysis [DiLella et al., Methods in Enzymology 152:199 (1987)]. The average molecular size of the isolated DNA was determined using field inversion gel electrophoresis [Carle et al., Science 232:65 (1986)] and ranged from about 500 kb to greater than 3 mb. DNA was digested with WJol under conditions recommended by the manufacturers and the digestion terminated by phenol/chloroform extraction. Following digestion, the DNA was analyzed on field inversion gels or 0.3% agarose gels to determine the average size of the digestion products. For the construction of genomic libraries in cosmid vector sCOS-1 genomic DNA was digested to an average size of 100 - 120 kb, and dephosphorylated with calf intestinal phosphatase. The genomic DNA was not size separated before cloning. Vector cloning arms were prepared by first digesting purified sCOS vector DNA with XJal followed by dephosphorylation with calf intestinal alkaline phosphatase. The reaction was terminated by phenol/chloroform extraction and the DNA collected by ethanol precipitation. The linearized, dephosphorylated vector DNA was then digested with BamHI, extracted with phenol/chloroform and stored at a concentration of 1 mg/ml in 20 mM TRIS-HCI, pH 6, 1 mM EDTA. Ligations were performed using 1 μg of vector arms and 50 ng to 3 μg of genomic DNA. Reactions were incubated with 2 Weiss Units of T4 DNA ligase and packaged using commercial in vitro packaging lysates. Bacteriophage lambda packaging extracts may contain significant amounts of EcoK restriction activity. To avoid the possibility that mammalian sequences containing an EcoK site might be underrepresented in the library, genomic libraries are prepared using in vitro packaging extracts which lack EcoK restriction activity (e.g. Gigapak-Gold; Stratagene Cloning Systems, San Diego, CA) . Cosmid libraries were plated directly on LB agar (LB media containing 1.2% Bacto-agar; autoclave) containing 25 μg/ml of kanamycin sulfate and libraries screened without further amplification [Evans et al., Methods in Enzymology 152:604 (1987)]. Libraries were stored as original non-amplified plate stocks in LB media (10 g Bacto-tryptone, 5 g yeast extract, 5 g NaCI per liter of water; autoclave) with 15% glycerol at a concentration of 2.2 x 10 bacteria/ml at -70°C. The cosmid library used in the study described in the examples consisted of 1.5 x 10 independent clones. Selection of Human Clones from a Somatic Cell Hybrid Genomic Library Cosmid li ■brari•es were plated on 570 cm2 LB agar trays at a density of 10 clones/cm , replica filters prepared and filters hybridized with human placenta DNA labeled with 32 P-dCTP to a specific activity of 10 8 cpm/μg. Under these hybridization conditions, no background hybridization was detected against cosmids carrying mouse genomic DNA. Cosmids containing human genomic DNA inserts were picked with toothpicks, rescreened by hybridization to 32P-labeled human DNA, and archived in 96-well microtitre plates containing LB media, 15% glycerol and 25 μg/ml kanamycin sulfate at -70°C. Individual clones isolated from cosmid libraries were routinely grown, replicated, and DNA prepared using standard round-bottom 96-well microtitre plates. Replica transfer of clones in 96-well microtitre plates and transfer from archived plates to screening filters was carried out using an aluminum "hedgehog" made from 3-mm diameter brass rods set in plastic block, as described by Coulson et al.. Supra (p. 7822), or a laboratory robot (Beckman Biomek 1000) . Plating and Screening Libraries For multiplex analysis, archived cosmids were inoculated on the surface of a nitrocellulose or nylon based filter in a matrix or "grid" pattern. The size and density of the "grid" was determined by the pattern of wells in a standard 96-well microtitre plate and, in the experiments described in the examples, a 36 x 36 matrix was used. Before applying bacterial culture, a matrix pattern prepared on paper was transferred directly to the filter membrane by passing the filter through a copying machine followed by autoclaving. The clones were allowed to grow on the surface of the filter at 37°C for 12 to 15 hours and bacterial DNA was fixed to the filter using a standard colony lysis procedure [Vogeli et al., Methods in Enzymology 152:407 (1987)]. RNA Probe Synthesis and Hybridization Reactions Cosmids were transferred from archives to fresh 96-well plates containing liquid LB media with 25 μg/ml kanamycin sulfate and incubated at 37°C in a humidified atmosphere for 6 to 10 hours. Supernatants from individual wells were pooled and DNA prepared using a previously described cosmid miniprep procedure [Evans et al.. Methods in Enzymology, Supra] . Cosmids constructed with vector sCOS-1, or one of its derivatives, yield up to 2 μg of DNA from a 300 μl culture. All probe synthesis and mapping reactions were carried out with DNA prepared from minilysates. In some cases, the pooled DNA was digested with a restriction endonuclease such as BatnHI or Hindlll prior to probe synthesis. RNA probes were synthesized using bacteriophage T3 or T7 polymerase (Stratagene Cloning Systems, San Diego, CA, USA) . Thus, cosmid DNA was prepared and 1-2 μg of the DNA was transcribed with T7 or T3 RNA polymerase in a 20 μl reaction, as described by Melton et al. (1984) Nucleic Acids Research 12.'7035-7054, using 50 μCi of [α- 32 P] UTP and 12 μM unlabeled UTP. 32P-UTP and polymerase reacti.ons were terminated by extraction with phenol and chloroform. 100 μl of blocking mixture (a mixture of sonicated human placenta DNA and cloned human repetitive sequences at a concentration of 1 mg/ml) was added, and the probe mixture was precipitated with ethanol. The nucleic acid was then resuspended in 20 μl of 5X saline sodium phosphate EDTA (SSPE) , 0.1% sodium dodecyl sulfate (SDS) , and prehybridized for 5 minutes at 42°C to saturate repetitive sequences which might be present in the probe. The probe was then added to a plastic bag containing a replica of the matrix filter and hybridization buffer (5X SSPE, 50% formamide, 0.2% SDS, IX Denhardt's solution (i.e., 0.2% Ficoll, 0.2% polyvinyl pyrrolidone, 0.2% bovine serum albumin; see Denhardt, Biochem. Biophys. Res. Commun. 3.:641 (1966)), and 20 μg/ml salmon sperm DNA) and the hybridization reaction carried out for 12 to 18 hours. Filters were washed in 0.1X SSPE, 0.1% SDS, at 65°C and exposed to X-ray film for 2 to 8 hours. End-specific Nucleotide Sequence Analysis Cosmids are transferred from archives to fresh 96-well plates containing liquid LB media with 25 μg/ml kanamycin sulfate and incubated at 37°C in a humidified atmosphere for 6 to 10 hours. DNA is prepared using a previously described cosmid miniprep procedure [Evans et al., Methods in Enzymology, Supra1. DNA from each cosmid is sequenced in a commercially available DNA Sequencer following the manufacturer's instructions. Restriction Enzyme Analysis Restriction enzyme analysis of isolated cosmids was carried out using DNA isolated from minilysates. Cosmid DNA was prepared from minilysates as follows: DNA was isolated from 1.5 ml cultures. A culture was inoculated with a single bacterial colony and incubated with vigorous shaking at 37°C for 6 hours. DNA was prepared using a modified boiling procedure [Evans et al.. Methods in Enzymology 152:604 (1987)]. Cells were collected by a brief (1 min.) centrifugation in a microcentrifuge and cells were resuspended in 300 μl of STET buffer (50 μM TRIS*HCI, pH 8.0, 8% sucrose, 5% Triton X-100 and 50 mM EDTA) . 20 μl of freshly prepared lysozyme (10 mg/ml) in STET buffer were added, the mixture vortexed and incubated in a boiling water bath for one minute. The solution was immediately centrifuged for 10 minutes in a microcentrifuge and the gelatinous pellet removed with a toothpick and discarded. 325 μl of isopropanol were added and the mixture incubated at room temperature for 5 minutes. The precipitated DNA was collected by centrifugation at room temperature in a microcentrifuge, the pellet dried and resuspended in water. DNA was digested to completion with NotI , digested partially with one or more enzymes (typically BamHI, EcoRI, Hindlll, SacII, PvuII, and Kpnl) , separated on an agarose gel, transferred to a nitrocellulose filter and hybri .di.zed wi.th 32P-labeled oligonucleotides recognizing the T3 or T7 bacteriophage promoters. T3 and T7 oligonucleotides (commercially available as sequencing primers, Stratagene Cloning Systems, San Diego, Ca, USA) were labeled using polynucleotide kinase and y- 2 P ATP to a specific activity of 2 x 10 cpm/μg. The labeled oligonucleotides were then hybridized to the filters in 6X saline sodium citrate (SSC) , 10% Denhardt's solution for 12 hours at 42°C and washed in 2X SSC for 10 minutes at 50°C. Filters were exposed to X-ray film for 20 minutes to 12 hours. The pattern of bands appearing on the autoradiograph could then be interpreted as indicating the distance from the cloning site to the restriction site, much as with the "cos"-mapping procedure of Rackwitz et al.. Gene 2_2:195 (1984). Alternatively, programmed automatic restriction enzyme digestions were carried out to completion in 96- well microtitre plates using a laboratory robot (Beckman Biomek 1000) . Data Analysis The resulting hybridization data were manually entered into a computer file and analyzed using computer programs written in Turbo Pascal (Borland International) running on Apple Macintosh II or Macintosh SE computers. One program "Multiplex-mapper" compared data sets from hybridization reactions using mixed probes, determined those clones which were identified by more than one probe mixture, and produced a list of linked clones. A second program, "Contig-maker" assembled the list of overlapping clones into potential contigs which could be analyzed in greater detail. In some cases, orientation and overlap of individual cosmid clones in a contig were confirmed by detailed restriction mapping and hybridization analysis of the individual cosmid clones. Although data analysis was performed using the above-mentioned computer programs, a person of ordinary skill in the art should have no difficulty in carrying out the comparison of sequence and/or hybridization data and assembling the overlapping clones into contigs using other software. Moreover, manual data comparison and contig making are also possible, though more laborious. EXAMPLE 2 Cosmid Multiplex Analysis of Human Chromosome llq The cosmid vector sCOS-1 (Figure 1) was used to prepare a genomic library from a somatic cell hybrid containing as its only human material DNA from the distal long arm of human chromosome 11, including llq21-llqter, in a mouse background [Maslen et al., Genomics 2.:66 (1988)]. The distal long arm of human chromosome 11 is of biological interest for a number of reasons. Like the major histocompatibility complex, the T cell receptor and immunoglobulin genes, and the IgK-CD8A-CD8B region of chromosome 2pl2, human chromosome llq23 contains a cluster of genes encoding proteins which are members of the immunoglobulin superfamily and are possibly important for cell-cell interactions in the immune and nervous systems including Thy-1, CD3, <_, epsilon, and N-CAM [Nguyen et al., J. Cell. Biol. i02.:711 (1986)]. Ilq23 is the location of genes in which defects may be responsible for ataxia telangiectasia [Gatti et al., Nature 336:577 (1988) ] and other hereditary disorders including multiple endocrine neoplasia type I [Larsson et al., Nature 332:85 (1988)], diabetes analogous to the NOD mouse [Prochazka et al., Science 237:286 (1987)] and others are also likely linked to markers on leukemias and pathognomonic for Ewing's sarcoma, peripheral neuroepithelioma and Askin's tumor [Griffin et al., Proc. Natl. Acad. Sci. USA 82:6122 (1986)]. The initial physical analysis of human chromosome 11 should allow eventual analysis of the genes associated with these phenomena and the underlying biology. A genomic library consisting of 1.2 x 10 individual members was prepared and cosmids containing only human DNA were selected from this library by screening with probes recognizing human, repetitive sequences. The proportion of human clones in this genomic library was 0.9%, indicating that the proportion of human chromosome 11 present in the somatic cell hybrid was about 27 mb, consistent with previous cytogenic and molecular characterization of this cell line [Maslen et al., Supra], 1296 clones were selected, archived in 96-well microtitre plates, and arranged on a nitrocellulose filter according to the columns and rows of a 36 x 36 matrix. Using probes recognizing many available DNA markers mapping to this chromosome, consmids containing the genes THY1 [van Rijs et al., Proc. Natl. Acad. Sci. USA 2 :5832 (1985)], T3D, T3E [Evans et al., Immunogen £8.:365 (1988)], ETS1 [Watson et al., Proc. Natl. Acad. Sci. USA 82:1792 (1986)], PBG [Wang et al., Proc. Natl. Acad. Sci. USA 78:5734 (1981)], PGR [Misrahi et al., Biochem. Biophys. Res. Commun. 143:740 (1987)], SRPR [Lauffer et al., Nature 318:334 (1985)], and APOA1 [Karathanasis et al. , Proc. Natl. Acad. Sci. USA J30.:6147 (1983)]. The identified genes and clone coordinates for DNA markers on human chromosome llq-llqter represented in the ordered cosmid set are shown in Table 2. Table 2 Identified genes and clone coordinates for DNA markers on human chromosome llq21 - llqter represented in the ordered cosmid set. cosmid clone marker coordinate (Y.X. 3,11 THY1 11,34 THY1 10.7 T3D (CO3y, S ) 7,13 ETS1 5,13 ETS1 4,22 PGR 13,27 APOA1 11,25 PBGD 12,19 D11S23 24,12 D11S24 24.8 SRPR 11,31 SRPR Additional available RFLP markers [Maslen et al.. Supra1 were also identified in this collection to allow eventual correlation of the emerging physical map of chromosome 11 with the linkage map. Groups of clones corresponding to 32 of the rows and 36 columns were pooled, and 68 hybridization reactions were carried out to replica filters according to the strategy outlined hereinbefore. Mixed probes detected a minimum of nine and a maximum of 46 cross- hybridizing unique clones on the filter matrix with each hybridization reaction using a pooled probe. When hybridization is carried out with a mixed probe consisting of RNA transcripts from cosmids of a row of the matrix, and a mixed probe representing a pool of all cosmids aligned along a column of the matrix, the cosmid clone which hybridizes with both mixed probes is linked to the clone located at the intersection of the row and column from which probe mixtures were prepared. To aid in the analysis of the data generated by this procedure, the Y and X coordinates of the cross-hybridizing clones are entered into a computer and matches identified using one of several computer programs. From this series of experiments, 1099 linked clones were detected from the hybridization of 36 pooled columns and 32 pooled rows of the matrix. Several of these overlapping clones were analyzed by restriction mapping to confirm that the clones indeed did overlap in the expected manner. Completeness of the Cosmid Multiplex Data From the list of linked clones produced by this multiplex technique, contigs were assembled either manually or through computer analysis of the data from the predicted hybridization linkage using mixed multiple RNA probes. Based on an initial analysis of the data using a simple algorithm for contig construction, 315 contigs were assembled from the 1099 linked clones determined from multiplex analysis. The size of the contigs ranged from 2 linked cosmids to 27 cosmids grouped into a contig extending over several hundred kb, with the majority of contigs consisting of between 2 and 5 cosmids. To confirm that these groupings reflected the true structure of the human chromosome, and not artifactual groupings due to random cross-hybridization, several of the contigs were restriction mapped in detail to determine the degree of overlap and establish a physical map. The restriction map of a representative contig assembled by this strategy is shown in Figure 5. Assessment of Progress Based on the assumption that the region of human chromosome 11 carried by the parent hybrid represents about 27 mb, the collection of 1296 cosmid clones analyzed here represents about 2 genome equivalents. It is also estimated that the minimal detectable overlap by hybridization analysis using end- specific RNA probes is about 200 nucleotides. If θ is the fraction of length of two clones which must be shared in order for overlap to be detected [Lander et al., Supra] , then the expected number of contigs consisting of at least two clones generated by the analysis of N cosmid clones is Ne -c(l-θ)_ Ne -2c(l-θ) wherein the redundancy of coverage, c = LN/G, where L is the length of the clone insert and G is the haploid genome length in base pairs. The minimum detectable overlap with end- specific RNA probes is θ=0.005. Approaching the theoretical limit of θ=0, a maximum of about 450 contigs would be expected to result after the analysis of one genome equivalent and about 260 contigs after the analysis of 2 genome equivalents. Thus the analysis of the clone set carried out here, generating 315 contigs after the analysis of about two genome equivalents, is in good agreement with theoretical predictions. The main advantage of the current strategy is that the analysis of 1296 clones required only 72 analytical reactions, rather than 1296. It was found that the prehybridization of the RNA probes with a high concentration of human repetitive sequences, as hereinabove described, was sufficient to completely block hybridization of most of these frequencies, and was sufficient for eliminating most of these artifactual linkages. However, the analysis of several large contigs mapping to human chromosome 11 generated by this analysis has revealed several cosmid clones which were included in a contig but which could not be substantiated based on the result of restriction mapping and hybridization analysis. This artifact may be the result of cryptic low-frequency repetitive or redundant sequences present in this region of the genome, or could be the result of genomic sequences which are unstable and deleted or rearrange when cloned in E. coli . Evidence for the later sequences, isolated through screening non-amplified cosmid libraries, has been found in the analysis of the human CD3 locus [Evans et al., Immunogen, Supra1. However, it should be noted that the multiplex technique of the present invention, when carried to completion using both T3 and T7 mixed RNA probes, generates data that is internally redundant in that both members of a linked pair should cross-hybridize with one another. Thus, further refinement of this approach should eliminate most serious artifacts arising during multiplex clone analysis. In this regard, the analysis disclosed in the present invention has generated a partially overlapping cosmid set which is estimated to include about 60% of the llq21-llqter region of human chromosome llq. The results of certain preliminary restriction enzyme analyses, further analysis of contigs and filling-in by traditional chromosome walking are in complete agreement with theoretical calculations of fingerprinting efficiency. A more complete analysis of this and other chromosome regions using a number of cosmids for 4 or more genome equivalents would be expected to result in near closure of the map. Using the technique of the present invention, this would require a collection of about 3600 cosmids and 120 T3 or T7 reactions/hybridizations rather than the 72 carried out in the present Example. In addition, the technique of the present invention is applicable for traditional chromosome "walking" to allow "filling-in" of gaps in a near complete map. Additional analysis of this cosmid set representing chromosome llq can be completed by automated restriction mapping. Analysis to date has revealed the presence of 177 potential "linking" clones, containing one or more WotI restriction sites, and 77 clones containing SacII sites indicative of hypomethylated CpG- rich islands. Forty of these cosmid clones contain clustered rare CpG rich restriction sites and can be identified unequivocally as hypomethylated islands. In addition, cosmid clones have recently proved very useful for in situ hybridization to metaphase or interphase chromosomes [Lichter et al., Proc. Natl. Acad. Sci. USA 5:9664 (1988)] and the identification of the cytogenic location of single-copy DNA sequences. These procedures potentially will allow ordering cosmid contigs with resolution of greater than 500 kb and, coupled with the strategy described here, provide a powerful mechanism for the constructions of physical maps of chromosomes. Still further analysis of this cosmid set representing chromosome llq can be carried out by direct automated DNA sequencing of the fragment ends as described above in the Detailed Description and in Examples 3 and 4. EXAMPLE 3 Sequence-Sampled Map of G. lamblia genome A 10.5 mb genome of Giardia lamblia (Fan et al., 1991, Nucleic Acids Res. 19:1905-1908) was cloned as a twenty-genome equivalent cosmid library. Five thousand cosmids can be mapped, end-oriented and end-sequenced generating 10000 ordered sequence fragments spaced, on average, every one kilobase. The determination of 500 bp of DNA sequence directly from each cosmid end results in an average spacing between islands of 0.5 kb. Cosmid clones A Giardia lamblia cosmid library was constructed in vector sCos-1. The library was prepared by partially digesting WB strain Giardia lamblia genomic DNA with Sau3A and cloning the digestion products into the BamHI site. From primary platings, 6,250 clones, representing about 2OX coverage were picked and individually archived in 96-well microtitre plates and stored as frozen glycerol stocks. Clone names are derived from the library designation (GR) followed by the plate number and well position (e.g., cGR-2bll denotes a cosmid located on plate 2 in well bll) . These were arrayed onto filters with a Biomek 1000 robot (Beckman Instruments, Fullerton, CA) and the DNA fixed onto the filters using well known methods for analysis by hybridization. A specific hybridization probe to the β- giardin region was used to detect sixteen overlapping cosmids and a random probe detected another 26 overlapping cosmids. These cosmids were subjected to a series of mapping and sequencing experiments. Automated DNA sample preparation Template cosmid DNA was prepared by an alkaline lysis procedure (Sambrook et al., supra.. or by using one of several DNA prep robots. Automated procedures used DNA prepared by an Autogen 540 DNA preparation robot (cycle 411) , which was subsequently digested for one hour with RNAse A (75 μg/ul) in a total volume of 23 μl , and then precipitated with ethanol. Overnight growth of cosmid clones in 5 mis of terrific broth (see, Sambrook et al., supra) with 10 μg/ml kanamyacin generally provided sufficient DNA for one or two sequencing reactions. Automated DNA sequencing DNA sequencing was carried out using primers complementary to the T3 or T7 polymerase promoter located in the cosmid vector flanking the insertion site. Template cosmid DNA was prepared by an Autogen 540 DNA preparation robot (cycle 411) . Automated sequencing reactions were carried out using dye labeled T3 or T7 oligonucleotide primers with reactions assembled and cycle sequenced using the Applied Biosystems (ABI, Foster City, CA) Catalyst 800 robot with DNA concentrations of -0.2 μg/μl. Sequence determination was carried out using the ABI 373A fluorescent sequencer. The labels (names) given to each of the DNA sequences determined from the cosmid clones are the clone names followed by -t or -u, denoting sequences from the T3 or T7 priming sites, respectively (e.g., cGR-19a9-u is the sequence of the T7 end of the cosmid clone found on plate 19 in position A9 in the GR library) . The increased organization permitted by the new nomenclature system used in Examples 3 and 4 will facilitate the large scale physical mapping strategies described herein. Contig construction and physical mapping Identification of cosmids from the .-giardin genomic region was accomplished using filters representing five-fold of the twenty-fold genomic library. Bacterial clones were stamped into a grid pattern with a Biomek 1000 robot using S & S Nytran filters with a 0.4 μm pore size and fixed to the filters using standard fixation procedures. A probe of approximately 1 kb recognizing ?-giardin genomic DNA was generated by PCR with the oligonucleotides GGTCAAGCTCAGCAACATGA (SEQ ID NO:l) and TGCTTTGTGACCATCGAGAG (SEQ ID NO:2) with standard amplification conditions and an annealing temperature of 60° C. Similarly, a random probe was generated as a 1.5 kb product with the primers CAGCAGATGGTCAAGCAAAA (SEQ ID NO:3) and ACTCCTGACACCACCACCTC (SEQ ID NO:4) . Physical and sequence maps were constructed by full digestion of each cosmid with the restriction enzymes Wcol and Bglll. Restriction fragments were separated on a 0.4% high strength agarose gel (0.5X TBE; see Sambrook et al., supra, run for 24 hours at 22v in a 20 by 20 cm gel apparatus. The Wcol and Bglll restriction enzymes digest the vector opposite the cloning site generating genomic fragments. Each end of the genomic insert was detected as a vector/genomic chimera by hybridization with probes flanking the T3 and T7 promoter sites of sCos-1. The 1046 bp t3 probe was amplified from sCos-1 with the primers (5- to 3*) TCGCTCACTGACTCGCTG (SEQ ID NO:5) and AGCCCTCCCGTATCGTAGTT (SEQ ID NO:6), and the 1004 bp T7 probe with the primers CTTGAGAGCCTTCAACCCAG (SEQ ID NO:7) and AACTGGGCGGAGTTAGGG (SEQ ID NO:8) with an annealing temperature of 60°C. and the standard conditions described above. The T7 probe was labeled by random priming with S dATP and the t3 probe with 33 P dATP for dual-label hybridizations. Maps were constructed by determining an order of fragments with no gaps with the GRAM program [see, e.g., Soderlund et al., in Proceedings of the 26th Hawaii International Conference on System Sciences : Biotechnology Computing. (ed. Hunter, L.) 620-630 (CA: IEEE Computer Society Press, 1993)]. The mapping strategy consisted of digesting each cosmid with two different restriction enzymes and precisely determining the number and sizes of the various products. The enzymes Wcol and Bglll were chosen for mapping because their recognition sites have different numbers of G/C bases and their sites are located several kb from the vector cloning site. Fragment sizes were estimated with the GelReader program of T. Redman (National Center for Supercomputer Applications, Urbana, Illinois) and maps constructed with a modified version of GRAM, which takes the end fragments into account. Maps of oriented cosmids were generated by comparison of the two maps of restriction fragments (Figure 6) . The ends were placed by fit relative to the neighboring sites for 32 of the 42 cosmids while the remaining ten were determined by the concordance of the possible orientations of ends on both enzyme maps using neighboring known cosmid ends as anchors. One llh4 was equivocal and the FASTA analysis of the derived sequences showed that the t7 end matched the reverse complement of the 25h9 t3 sequence. Seguence Analysis The presence of repetitive sequences was determined using the program FASTA which compares all sequences to those previously determined supplemented with a comprehensive set of di- and tri-nucleotide repeats. A FASTA cutoff score of 100 was used to recognize repetitive sequences from background random matches. Similarities to known genes were identified with the BLAST program and the GenBank database. Amino acid comparisons were performed by translating DNA sequence fragments into all six potential reading frames and comparing translations to protein sequences in the non-redundant Swiss-Prot, GenPept or PIR database of the National Center for Biotechnology Information (Bethesda, MD) using the program BLASTX. The results of these various searches were evaluated numerically and by inspection (see, e.g.. Table 3). The data obtained as described herein, including DNA sequence file pointers, matches from sequence analysis and information about overlapping clones, were stored in a relational database. The sequences generated by automated fluorescent sequencing from cosmid ends have been deposited with GenBank and were not edited to remove unidentified bases or correct the sequence. Table 3 -Sequence searches performed with BLAST which produced likely, possible and interesting similarities to known sequences 3 with the sources of sequences, scores, percent identical residues and the length of amino acids matched. The highest scoring BLAST match is shown when more than one region or protein was similar to the unknown sequence. sequence similar to: score %JD length C 00 Likely H cGR-38b8-t* DNA similar lo 3" noncoding region surface antigen gene, G. lamblia (gb:Ll 1331) cGR-45h5-u protein kinase nekl, mouse (gb:S45828) cGR-41eil-u VSP1267 surface antigen, G. lamblia (sp:TSA4_GIALA) cGR-62el2-u protein kinase nekl, mouse (gb:S45828) X m oGR-15c7-ι b G2 specific proiein kinase, A. nidulans (sp:NIMA_ASPNl) H cGR-45h5-t surface protein 11, G. lamblia (gb:M95814) 30 C Possible cGR-16glO-u transcription factor Oct-1, mouse (sp:HMOl_MOUSE) en cGR-15c7-u MYB Protooncogene proiein, D. dis oidium (gb:S93742) 2 Searches presented were proiein sequence based with the exception of an identical nucleotide sequence found for cGR-38b8-L b The overlapping sequence determined from cGR-15c7-t and cGR-6a6-u were searched together as one TaWe 3 . Sequence searches performed with BLAST which produced likely, possible and interesting similarities to known sequences* w i th the sources of sequences, scores, percent identical residues and the length of amino acids matched. The highest scor i ng BLAST match is shown when more than one region or protein was similar to the unknown sequence. sequence similar to: score 9M0D length Interesting cGR-51 a9-u eukaryotic initiation factor 4E, mouse (sp:IF4E_M OUSE) cGR- 1 d7-t polyposis coli proiein DP 1 , human (gb: 73547) cGR-57alO-u ribonuclease inhibitor, rat (sp:R__N RAT) to I cGR-22b8-t dynein associated (glued) proiein, D. melanogaster (sp:DY 15_DROME) m rn cGR-25h9-t cosmid F09G8, C. elegans (gb:Ll 1247) cGR-2g8-u ORF in Ihr4-sup61 intergenic region, S. cerevisiae (sp:YCV0_ YEAST) "30 C cGR- J 9g 30-u M5 protein precursor, S. pyogents (sp:M5_STRPY) r- rπ cGR-16g8-u zinc f ger proteins, X. laevis (pir:S06546) rυ σ. cGR-16gl0-t lamin B2, chicken (sp:LA_v!2_CHIC ) cGR-41f7-u ankyrin 2, brain, human (piπS 14569) - Searches presenled were protein sequence based with the exception of an identical nucleotide sequence found for cGR-38b8-L ~ The overlapping sequence determined from cGR-15c7-t and cGR-6a6-u were searched together as one Sequences derived from cosmid ends were analyzed for the presence of repetitive sequences, simple sequence repeats and similarities to known genes (Figure 7) . A number of genes were detected during the characterization of sequences as described herein. There were three protein inases and a surface antigen gene in the random genetic region; no additional genes were detected near the 3-giardin gene. Additional genes could likely be determined with gene prediction programs developed for these applications such as GRAIL. Computer-aided primer pair design One advantage of mapping and sequence sampling is future independence from clone libraries through PCR amplification. This advantage was demonstrated within the random region contig. The selection of primer pairs for PCR analysis was carried out using the PRIMER program provided by E. Lander (MIT) for each of the cosmid- derived end-sequences that were determined. Analysis was done in batch processing mode on a Sun workstation specifying an annealing temperature of 60°C and a primer length of 18 - 22 nt. Modifications of these parameters (oligonucleotide length 25 nt and annealing temperatures of 55°C for AT-rich sequences and annealing temperatures to 65 β C or greater for GC-rich sequences) generally allowed production of a suitable primer. Primers were produced commercially by Genset, Inc. (Paris, France) . Predicted STS primers were tested by PCR amplification in a 30 1 reaction volume containing: 10 mM Tris (pH 8.8), 50 mM KCI, 1.5 mM MgCl 2 , 0.001% gelatin, 200 M each dNTP, 100 ng genomic DNA and 1.5 units of Taq DNA polymerase. Initial PCR conditions were: denaturation at 93°C for 2 minutes; 35 cycles of 30 seconds each at 94°C; annealing for one minute at the predicted annealing temperature, and 30 seconds at 72°C; followed by a final extension at 72°C for five minutes. The probes were labeled by random priming with a P dCTP and sCos-l DNA was labeled with 35 S dATP to identify specific clones in the hybridization process. The ordered sequences were then checked by PCR amplification demonstrating amplification of a specific region of interest independent of the cosmid libraries. Products were obtained for most (>85%) of the neighboring sequences. Some failures, possibly due to incorrect primer sequences and large distances between primers were not analyzed further within the random contig region. In summary, two regions of the Giardia lamblia genome were mapped and sequences sampled from within those areas of approximately 160 kb (Figures 6A and 6B) . The sequenced cosmid ends encompass approximately 15% of this total region. The average sequence read was 347 bp which does not differ from the median (353 bp) and has a standard deviation of 39 bp. Eight cases of cosmids ending at exactly the same Sau3A sites were observed and in four cases cosmid ends were close enough to one another to form small sequence contigs. Overall, the median spacing between cosmid ends was 1.25 kb with an average spacing of 2.0 kb, suggesting that the relatively rare events of cloning from defined restriction sites follows a poisson process suggested by visual inspection of Figure 8. These are underestimates of sequence overlaps and identical ends since cosmids which intrude into the contig without encompassing the region detected with the hybridization probe were not included in the contig. Full characterization of the region would nearly double the amount sequenced to approximately 30%. The large number of identical ends compared to overlapping sequence contigs suggests that the practical limit for constructing a library with one restriction enzyme for partial digests is below twenty fold and perhaps as low as five to ten fold in the sequence sampling strategy. EXAMPLE 4 Preparation of a Chromosome-11 Sequence Sampled Map Cosmid libraries Two chromosome 11-specific cosmid libraries were constructed in vector sCos-1. Cosmids denoted llq were isolated from a library prepared from hybrid TG5D1-1 representing chromosome llql3-llqter (Evans and Lewis, 1989, Proc. Natl. Acad. Sci.. USA. 86:5030-5034). TG5D1-1 is a Friend cell line derived from somatic cell hybrid 5D1 that carried an intact human X chromosome 11 [Pyati et al., Proc. Natl. Acad. Sci USA 77:3435 (1980)], and was selected for the loss of the entire X chromosome and most of chromosome 11. TG5D1-1 contains the distal portion of chromosome 11 as the only human material in a mouse genomic background [Maslen et al., Genomics 2:66 (1988)]. Cytogenetic and molecular analysis indicates that the amount of human DNA represented about 1% of the mouse genomic background [Maslen et al., Supra] . Cosmids denoted SRL were isolated from a flow sorted chromosome 11-specific library prepared from somatic cell hybrid Jl (described in Kao et al., 1977, Somatic. Cell. Genet.. 3:421-429). The SRL library was prepared from 100 ng of flow purified chromosome 11 at the Los Alamos National Laboratory (L. Deaven and J. Longmire) as part of the National Gene Library Project and represented 125X coverage of chromosome 11. Approximately 17,000 clones, representing about 5X coverage, were picked from primary platings and individually archived in 96-well microtitre plates for use in this example. Characterization of this library revealed that about 4% consist of non-human inserts. Clone names are derived from the library designation followed by the plate number and well position (e.g., cllq-2bll denotes a cosmid from the cllq cosmid library located on plate 2 in well bll) . Cosmid end sequencing To generate sequence data from the cosmids, DNA sequencing was carried out using primers complementary to the T3 or T7 polymerase promoter located in the cosmid vector flanking the insertion site. Template cosmid DNA was prepared by an alkaline lysis procedure (Sambrook et al., supra) . or by using one of several DNA prep robots. Automated procedures used DNA prepared by an Autogen 540 DNA preparation robot (cycle 411) , which was subsequently digested for one hour with RNAse A (75 μq/μl) in a total volume of 23 μl, and then precipitated with ethanol. Overnight growth of cosmid clones in 5 mis of terrific broth (Sambrook et al., supra) with 10 ug/ml kanamyacin generally provided sufficient DNA for one or two sequencing reactions. Alternatively, DNA template preparations were done using the custom "Prepper, Ph.D." DNA preparation robot developed by H. R. Garner (Garner et al., Sci. Computing and Automation. March/1993, 61- 68) . Some of the initial sequences in this example were determined manually using Sequenase kits with dideoxy terminators (U. S. Biochemicals) . Automated sequencing reactions were carried out using dye labeled T3 or T7 oligonucleotide primers with reactions assembled and cycle sequenced using the Applied Biosystems (ABI) Catalyst 800 robot with DNA concentrations of ~0.2 μg/μl. Automated DNA sequencing was carried out using the ABI 373A sequencer. The names of DNA sequences determined from the cosmid clones are the clone names followed by -t or -u, denoting sequences from the t3 or t7 priming sites, respectively. (e.g., cllq-2bll-u is the sequence of the t7 end of cosmid 2bll from library llq) . A sample of 371 DNA sequence fragments were determined by automated sequencing from cosmid ends. Forty-nine sequences were not used for STS production in this example and were placed in reserve should additional STSs be needed. Extensive regions of repetitive sequence which made PCR primer prediction unlikely occurred in 14% (45/322) of those analyzed (Table 4) . The remaining 277 sequences (86%) were used for STS primer prediction by computer analysis and oligonucleotide primers were synthesized and tested. STS MAP OF CHROMOSOME 11 TABLE 4 Analysis of 322 cosmid-derived sequences for STS production percentage Fate of sequences number of total Not suitable for primer prediction 45 14% *_. t t U) X m failed STS production 65 20% successful STSs production 212 66% _30 C STSs mapped to a region 187 58% Note: Cosmid sequences were determined by direct priming of cosmid templates using oligonucleotides complementary to the T3 or T7 polymerase promoters present in the vector. STS primers were predicted using the PRIMER program. The sequences generated by automated fluorescent sequencing from cosmid ends have been deposited with GenBank and were not edited to remove unidentified bases or correct the sequence. The sequence information was not corrected before analysis so that the Primer program would not predict primers in regions of questionable accuracy. Consequently, some of the alignments showing similarity between known protein sequences and translated chromosome 11 specific sequences contain Xs at unknown and stop codons which could be due to errors in the sequence or translating beyond exons into introns. Computer-aided primer pair design The selection of primer pairs for STS analysis was carried out using the Primer program available from E. Lander (MIT) . Analysis was done in batch processing mode on a Sun workstation specifying an annealing temperature of 60° C and a primer length of 18 - 22 nt. Modifications of these parameters (oligonucleotide length 25 nt and annealing temperatures 55"C for AT-rich sequences and annealing temperatures to 65°C or greater for GC-rich sequences) generally allowed production of a suitable primer set. DNA sequences that contained extensive regions of repetitive sequence so that primers could not be designed to generate products of at least 120 bp were not utilized. Primers were synthesized using an ABI PCR-Mate oligonucleotide synthesizer or produced commercially by Genset, Inc. (Paris, France) . Of this collection of primers, 77% (212/277) generated PCR amplification products from human genomic DNA and Jl hybrid DNA without producing products from yeast genomic DNA and are suitable for STS content mapping using YAC libraries. Localization of 88% of these new STS markers (188/212) was carried out by hybrid analysis, FISH or a combination of both. For comparison, nearly the same success rate occurred in predicting primer sets from sequences retrieved from GenBank. In some cases primer sets derived from the cSRL library which failed to amplify human DNA clearly generated a product from hamster — the Jl cell line host (Table 5) . STS MAP OF CHROMOSOME 11 Table 5 Hybrid cell lines containing specific regions of human chromosome 11 C DO C H O * - m X m m 3 C r en Note: The species from which cell lines were derived and a reference is indicatred above. STS markers generated from mapped cosmids were supplemented with STSs corresponding to almost all of the cloned genes on chromosome 11 to allow for eventual production of a complete chromosome map. Genes mapped to chromosome 11 were identified by searching the Genome Data Base (GDB; Pearson et al, 1992, Nucleic Acids Res.. 20:2201-2206), On-line Mendelian Inheritance in Man (OMIM) , GenBank, Medline, and the like. The DNA sequences were retrieved from GenBank when available and STS primers designed using PRIMER. Whenever possible, chromosome 11 genomic DNA sequence was used for STS production and primer pairs were designed to generate products of 400 to 1000 base pairs. When genomic DNA sequence was not available, the generally intron-free 3' untranslated region of chromosome 11 cDNAs was chosen. Where these were not available, cDNA coding regions were utilized and the predicted amplification product sizes were limited to 150-250 bp in order to minimize the chances of including an intron in the target region. In the overall design of STS primer sets, predicted product lengths were intentionally varied over the range 150 to 1000 bp to allow for eventual multiplex screening of YAC libraries. Characterization of chromosome 11-specific STSs Predicted STS primers were tested by PCR amplification (Saiki et al., 1988, Science, 239:487-491) in a 30 μl reaction volume containing: 10 mM Tris (pH 8.8), 50 mM KCI, 1.5 mM MgC12, 0.001% gelatin, 200 μM each dNTP, 100 ng template DNA and 1.5 units of Taq DNA polymerase. Initial PCR conditions were: denaturation at 93°C for 2 minutes; 35 cycles of 30 seconds each at 94°C; annealing for one minute at the predicted annealing temperature, and 30 seconds at 72"C; followed by a final extension at 72°C for five minutes. Initially, three different concentrations of primers (833, 500 and 250 nM) were tested for amplification using human genomic DNA. An optimal concentration and temperature were determined for the amplification of the following test DNA samples: human genomic DNA, DNA from the hybrid cell line Jl containing human chromosome 11 in a CHO cell background, hamster genomic DNA, mouse genomic DNA and yeast DNA. Those primer sets that produced an amplification product of the expected size from human genomic DNA and Jl DNA were characterized further. Most of the STSs generated have a narrow range of annealing temperatures from 56°C to 60°C and relatively short primer lengths of around 20 nucleotides. Physical Mapping by fluorescence in situ hybridization .FISH) To map the genomic location of each STS, in situ hybridization using cosmid DNA was carried out on etaphases prepared from human fibroblasts (CRL1634; Human Genetic Mutant Cell Repository, Camden, New Jersey) using well-known methods (see, e.g., Giovannini et al., 1993, Cvtogenet. Cell. Genet.. 63:62). Chromosomal localization was determined as chromosome 11 FLpter (fractional chromosomal length from llpter) and cytogenetic band position was extrapolated from the coincidence of FLpter values to cytogenetic bands on the chromosome ideogram. Somatic cell hybrid analysis Additional localization of some cosmid sequences, and localization of STSs produced from gene sequences, were carried out using a panel of hybrid cell lines set forth in Table 5 containing part or all of human chromosome 11 in mouse or hamster genomic backgrounds. Analyses of the PCR amplification of STSs from DNA isolated from this set of hybrids mapped the STSs to eight distinct regions, or bins, on chromosome 11 (Figure 10) . The whole set of 370 STS markers, associated with known genes or chromosome-specific cosmid clones, was regionally mapped to chromosome 11. Some of the map locations of these cosmids had been reported previously, though not associated with STS identifiers of the present invention. Of the 370 STSs generated, 335 were successfully mapped to specific regions of human chromosome 11 (Table 6) using FISH or through the PCR analysis of a somatic cell mapping panel. Mapping information for some markers was available from GDB and was confirmed employing the techniques described herein based upon hybrid analysis. Consistent results were found in all cases when mapping information on many markers was determined using both FISH and hybrid analysis. In general, the set of standard STSs derived in this example appear to be uniformly distributed throughout the chromosome and include markers for most of the significant mapping landmarks. Chromosome 11 was divided into twenty regions and the average distribution of STSs along chromosome 11 was assessed. The average number of STSs per region was 17 with a standard deviation of 4.5. The most over-represented region was FLpter 0.85 to 1 (llq23.3 - q25) while 0.3 to 0.45 (llpl2 - ql2.1) and 0.6 to 0.85 (llql4.1 - q23.2) contained proportionally fewer STSs. The relative precision of the mapping information can be estimated from the distribution of FLpter ranges of the 335 mapped STSs. Twenty-four percent of the markers had an FLpter range of less than 0.05, 55% less than 0.1 and 83% less than 0.2. STS MAP or CHROMOSOME Table 6 Chromosome 11 specific STSs, mapping positions, technique 9 , product size, primer names, se uences, concentrations and annealin tem eratures - 0 I H m IΛ X 73 C ι— m tv_» STS MAP on CHROMOSOME I I Map ProducI Primer [pπmerj Annealing lech, size (bp) name Primer sequence (nM) temp (°C) be 177 55 101 58 O 216 51 o be 256 <") * *. 56 t* I 257 51 c 228 58 30 C 150 52 m 161 56 207 56 190 58 STS MAP OF CHROMOSOME 11 STS Name FLpter range Map ProducI Primer (primer) Annealing Locus Name (If different) band(s) tech. size (bp) name Primer sequence (nM) temp.( Q C) D11S783 Cl1q-Bd10 50 D11S784 c11q-8d11 58 Λ D11S1008 c11q-8h4 56 DO IΛ H D11S1009 c11q-9a1 56 H C H o** D11S1001 c11q-10d7 65 IΛ X m D11S1011 cS L-1a2 56 73 D11S1010 cSRL-1a12 56 C D11S1013 cSRL-1b2 56 D11S1014 cSRL-1b4 58 D11S1015 cSRL-1b8 60 STS MAP OF CHROMOSOME 1 1 (primer) Annealing STS Name FLpler range Map Product Primer Locus Name (If different) band(s) tech. size (bp) name Primer sequence (nM) lemp.(°C) D11 S1012 cSRL-1b11 0.40-0.55 151 cSRL-lbl l-uA 58 11p11.11-q13.3 cSRL-1b11-uZ D11S1016 cSRL-1c5 0.79-0.85 αb 185 cSRL-1c5-IA 58 11q23.1-q23.3 cSRL-1c5-IZ c IΛ D11S1017 cSRL-1cB 0.93-0.93 αb 175 cSRL-1c8-IA 58 1 1q24.1 cSRL-1c8-lZ CO IΛ H D11S1018 cSRL-1d4 0.24-0.33 211 cSRL-1d4-uA 58 H 11p13-p11.2 cSRL-1d4-uZ C a** D11S1019 cSRL-1d5 0.05-0.24 -P- 141 cSRL-1d5-uA 56 11p15.4-p13 cSRL-1d5-uZ IΛ X D11S1020 cSRL-1d6 0.05-0.12 αb 241 cSRL-1 d6-IA 58 11p15.4-p15.1 cSRL-1d6-IZ 73 D11S1022 cSRL-115 0.33-0.50 134 cSRL-1f5-uB 56 c 11p11.2-q13.1 cSRL-1f5-uZ D11 S1023 CSRL-K8 0.85-0.93 215 cSRL-K8-uA 58 11q23.3-q24.1 cSRL-118-uZ D11S1021 cSRL-1112 0.00-0.05 157 cSRL-1f12-uA 57 11p15.5-p15.4 cSRL-K12-uZ D11 S1024 cSRL-1g5 nd 219 cSRL-1g5-uB 52 cSRL-1g5-uY D11S1025 cSRL-1g8 nd 155 cSRL-1g8-uB 54 cSRL-1g8-uY STS MAIΌFCII OMO.SΌMΠ I I STS Name FLpler range Map ProducI Piimer [piimβi] Annealing Locus Name (if dillerenl) band(s) loch, size (bp) name Primor sequence lernp.( O C) D11SI026 cSRL-1g9 56 c tΛ D11S1C27 cSRL-lhδ 50 CO _- D11S1028 cSRL-1un 58 H C H m D11S1C29 cSRL-2a2 56 wi X D11S1030 cSRL-2b3 56 H 30 D11S1031 cSRL-2d 58 D11S1033 cSRL-2c2 56 D11S1034 cSRL-2c3 56 D11S1035 cSRL-2c4 56 D11S1036 cSRL-2c5 56 STS MAP OP CHROMOSOME 1 I STS Name FLpler range Map Product Primer (primer] Annealing Locus Name (If different) band(s) lech, size (bp) name Primer sequence (nM) lemp.( Q C) D11S1037 cSRL-2c7 56 D11S1044 cSRL-2β1 56 DI1S1040 cSRL-2o2 50 D11S1049 cSRL-2o3 56 STS MAI-OI ; αiHOMO.SOMI: I I STS Name FLpler range Map ProducI Primer [primer) Annealing Locus Name (if dilferenl) band(s) lach. size (bp) name Piimor soqiionco (nM) lomp.( Q C) D11S1050 cSRL-2e4 56 D11S1051 cSRL-2o6 56 c (Λ DI1S1052 cSRL-2o7 56 00 D11S1053 cSRL-2o9 56 c H m DI1S1045 cSRL-2e10 56 tΛ X m D11S1046 cSRL-2e11 53 H 50 C D11S1047 cSRL-2β12 50 D11S1056 cSRL-212 56 D11S1057 cSRL-213 56 D11S1058 cSRL-214 56 STS MAPOPCIfROMOSOME 11 STS Name FLpler range Map ProducI Primer Locus Name (If differenl) band(s) lech, size (bp) name Primer sequence D11S1059 cSRL-215 0.05-0.24 256 cSRL-215-IA TA1TTCTCTTTCCATCCCTCC 11p15.4-p13 cSRL-215-tZ TTTACTAACCCTCAUCCCACC DI1SI0G4 cSrtL-2110 nd cSnL-2M0-iΛ TAACACΛCCAΛCAr.CAACCC CSRL-2110-IZ ATClCCACAGTCTGCACCπ DI1S1055 cSRL-2111 nd 267 cSnL-2111-IA AGCTTΠTTCATCTTΓGCTGC CSRL-2111-IZ CCAACTCCAACAAAACCAAA DUSI061 cSRL-2g4 0.93-1.00 b 267 cSnL-2g4-IA ACACTCCCTGCCAAAGATC 11q24.1-q25 cSRL-2g4-lZ AATTTCA CTrmrcm CC D11SI0C2 cSRL*2g6 0.05-0.24 b 224 cSRL-2g6lA CCCTCAAGCCTGCATΓCTAG < IΛ 11p15.4p13 cSRL-2g6-lZ AACAACAGTTTCCKCAAGCC X DUS1063 cSRL-2g7 0.6O-0.B5 b 151 cSRL-2g7-tΛ ATCACCATCTTTTTCTCCTACA 11q14.1-q23.3 cSRL-2g7-(Z ACCCAACCAACACΠAAATTGA 70 C D11S1064 cSRL-2g9 0.05-0.24 b 154 cSRL-2g9*lA TCCACCTCACATGACTCTCC i 11p15.4*p13 cSRL*2g9-tZ CCACTCCAAAAACACCCTAA m- N» D11S1060 cSRL-2g11 0.05-0.24 b 198 cSRL*2g11-IΛ TTTCπCTTTAAαCACCCCG en 11p15.4-p13 cSRL*2g11*lZ CCCCTCATCTCACACΠCCA D11S1067 cSRL-2h2 0.50-0.85 b 2G4 cSnL*2h2-tA UCΛACCCCTCTGTTACCTCC 11q13.1-q23.3 cSRL-2h2-lZ AAAACCACAATCCCATCACA D11S1OG0 cSRL-2h9 0.05-0.24 b 204 cSRL-2lι9-IΛ ACCCTCCCTCATCAΠTGC 11p15.4-p13 cSRL*2h9-lZ ACTCCΠTTGCACATCTACCC DI1S10G5 cSRL-2h10 0.6 -0.05 b 302 cSRL-2h10-IΛ ACACCTACCCTCCTTACCArrC 11q14.1-q23.3 cSRL-2h10-lZ TCATTCCTCACTCTCAATCCA STS MAP OPCIIROMOSOMI* 1 I STS Name FLpter range Map ProducI Primor Iprimer] Annealing Locus Name (If diflerenl) band(s) lech, size (bp) name Piimor sequβnco (nM) lomp.(°C) D11S1066 cSRL-2h11 56 Λ D11S1069 cSRL-3a1 56 3 Λ D11S1070 cSRL-3a3 56 D11S1071 cSRL-3a4 56 Λ r D11S1072 cSRL-3a6 56 3 D11S1073 cSRL-3a7 56 r- m D11S1074 cSRL-3a8 56 t a. D11S1075 cSRL-3a9 56 D11S1076 cSRL bl 57 D11S1080 cSRL-3b2 56 60 STS MAP OP CHROMOSOME 1 1 IΛ c 00 t_Λ H H C H m IΛ σ -_Z m 71 C m ■STS MAI'OPCIIUOMOSOMP. I f STS Name FLpler ranye Map Product Primer [prime * ] Annoaling Locus Name (II dillerβnl) band(s) lech, size (bp) name Primer sβqcionce (nM) lemp.(°C) D11S1086 cSRL-3d12 56 D11S1090 cSRL-3o1 56 IΛ c 00 IΛ D11S1093 cSRL-3e2 56 H D11S1094 cSRL-3e4 56 IΛ X D11S1095 cSRL-3o5 56 D11S1096 cSRL-3o8 56 513 O11S1091 cSRL-3e10 56 to en D11S1092 cSRL-3e12 56 D11S1099 cSRL-3h2 56 D11S1100 cSRL-3h3 56 D11S1101 cSRL-3h4 0.50-0.85 b 250 cSRL-3h4-tA 56 11q13.1-q23.3 cSRL-3h4-lZ D11S1102 cSRL-3h6 0.24-0.33 b 159 cSRL-3h6 tΛ 56 11p13 plt.2 cSRL-3l*612 0.24-0.33 b 252 cSRL-3h7-lA 56 11p13-p11.2 cSRL-3b7-l2 0.60-0.85 b 255 cSRL-3hBtA 56 11q14.1-q23.3 cSRL-3hB 12 nd IBS cSRL-3h10-IΛ 56 cSRL*3h10-l2 0.05-0.24 b 245 cSRL-3h1l-IA 56 11p15.4p13 cSRL-3h11-l2 73 C D11SI105 cSRL-4a4 0.50-0.60 b 157 cSRL-4a4 IΛ 56 11q13.1-q14.1 cSRL-4a4-l2 σ. D11S1106 cSRL-4a6 0.05-0.24 b 1G5 cSRL-4nGlΛ 56 11p15.4-p13 cSnL*4aG-l2 D11S1107 cSRL-4a7 0.24-0.33 b 190 cSRL-4a7-LA 56 11p13-p11.2 cSRL*4n7-tZ D11S1108 cSRL-4a9 O.b -0.93 b 150 cSRL-4o9tΛ 56 11q23.3-q24.1 cSRL-4a9-l2 D11S1109 cSRI-4b1 0.50-0.G0 255 cSRL-4b1 tΛ 56 11q13.1-q14.1 cSRL-4b1-t2 STS MAP OF CHROMOSOME 11 STS Name FLpler range Map Product Primer [primer] Annealing Locus Name (if different) ba-d(s) tech. size (bp) name Primor soquence (nM) lamp.(°C) D11S1112 cSRL-4b3 56 CΛ c D11S1113 cSRL-4b4 56 00 CΛ D11S1114 cSRL-4b5 56 -i c D11S1115 cSRL-4b6 56 IΛ X D11S1116 cSRL-4b7 56 m H 30 D11S1117 cSRL-4b8 52 C D11S1110 cSRL-4b10 56 CΛ D11S1111 cSRL-4b12 58 D11S1120 cSRL-4c2 56 D11S1121 cSRL-4c3 56 STS MAP OP CHROMOSOME 11 STS Name FLpter range Map Product Primer Iprimer) Annealing Locus Name (If differenl) band(s) tech. size (bp) name Primer sequence (nM) temp.( Q C) D11S1122 cSRL-4c4 56 D1IS1123 cSRL-4c6 56 IΛ D11S1124 cSRL-4cB 56 c DO D11S111B cSRL-4c10 H C H D11S1119 cSRL-4c11 CΛ J X DIISI125 cSRL-4dl m 56 m H Dl 1 S1127 cSRL-4d4 58 D11S1128 cSRL-4d6 56 t σ. DI1S1129 cSRL-4d7 56 011S1126 cSRL-4d10 56 D11S1130 cSRL-4e1 56 ■STS MA OF HUOMOSOMI: I I STS Name FLpler range Map Product Primer [primer] Annealing Locus Name (if differenl) band(s) lech, size (bp) name Piimor soquence tomp.(°C) D11S1131 cSRL-4e3 56 D11S1132 cSRL-4o4 56 Λ 00 CΛ D11S1133 cSRL~4o5 56 c 011S1134 cSRL-4β6 56 CΛ X 011S1135 cSRL-4β8 54 m m D11S1136 cSRL-4β9 56 73 C D11S1138 cSRL-412 54 CΛ D11S1139 cSRL-413 54 D11S1140 cSRL-416 D11SI141 CSRL-4IB 54 STS MA OP H OMOSOMΠ 11 STS Name FLpler range Map Product Primer [primer] Annealing Locus Name (II dilferent) bn-d(s) tech. size (bp) name Pdmor soquonce (nM) lemp.(°C) D11S1142 cSRI-419 54 D11S1137 cSRL-4112 54 CΛ c D11S1143 cSRL-4g1 54 00 CΛ D11S1146 cSRL-4g3 56 c H D11S1147 cSRL-4g4 56 CΛ X 011S114B cSRL-4gB 56 73 C D11S1149 cSRL-4g9 56 m D11S1144 cSRL-4g10 56 D1151145 cSRL-4g11 54 DI151152 cSRL-4h5 56 D11S1153 cSRL-4h8 56 STS MAI'OPCHUOMOSOMΠ 11 STS Name FLpler range Map ProducI Primer [primor] Annoaling Locus Name (II dillerenl) band(s) loch, size (bp) name Piimor sequence lemp.(°C) D11S1150 cSRL-4h11 56 D11S1151 cSRL-4h12 56 CΛ c 00 CΛ D11S1154 cSRL-5a1 56 H C D11S1157 H cSRL-5a2 56 m CΛ X D11S1158 cSRL-5a3 56 D11S1159 cSRL-5a4 56 73 C D11S1160 cSRL-5a5 56 ro DI1S1161 cSRL-5a6 52 D11S1162 cSRL-5a7 56 D1ISI163 cSRL-5a9 56 STS MΛPOI-Ci OMOSOMF. I 1 "" STS Nome FLpler range Hap ProducI Primer [pii er] Annealing Locus Name (II different) ba-d(s) lech, size (bp) name Primor soquence lemp.(°C) D11S1155 cSRL-5a10 0.24-0.33 285 56 11p13-p11.2 D11S1156 cSRL-5a11 0.50-0.60 290 56 11q13.1-q14.1 CΛ c D11S1164 cSRL-5b1 0.93-1.00 198 56 00 11q24.1-q25 D11S1165 cSRL-5b7 0.05-0.24 151 56 H C 11p15.4-p13 H D11S11G6 cSRL-5bβ 0.05-0.24 150 56 CΛ 11p15.4-p13 00 X D11S1169 cSRI-5c2 0.50-0.85 258 56 11q13.1*q23.3 30 c Dl 151170 cSRL-5c3 0.05-0.24 278 58 11p15.4-p13 ro DI1S1171 cSRL-5c5 0.24-0.33 307 56 σ-i 11"13-p11.2 D11SH72 cSRL-5cB 0.00-0.05 15Σ 56 11p15.5p15.4 D1IS1173 cSRL-5c9 0.60-0.85 242 56 11q14.1-q23.3 DUS1167 cSRI-5c10 0.60-0.B5 2GI 56 11q14.1-q23.3 STS MΛ OI-ΓHKOMOSOMI; I l STS Name FLpler range Map Product Primer [primer] Annealing Locus Name (II dilferenl) band(s) tech. size (bp) name Piimor soquonco (nM) lemp.(°C) D11S1168 cSRL-5c11 56 D11S1176 cSRL-5d2 58 CΛ c 00 CΛ D11S1177 cSRL-5d3 56 H H C D11S117B cSRL-5d5 56 H CΛ D11S1179 cSRL-5d6 X 56 m o m H D11S1180 cSRL-5d7 56 P0 c r- D11S11B1 cSRL-5d8 56 m ro σι D11S1182 cSRL-5d9 5B D11S1174 cSRL-5d10 56 D11S1175 cSRL-5d11 58 STS MAP OP CHROMOSOME 11 STS Name FLpter range Map ProducI Primer [primer] Annealing Locus Name (If different) ba ** d(s) tech. size (bp) name Piimor soquonco (nM) lemp.(°C) D11S11B3 cSRL-5e1 Dl 1S1 IBS cSRL-5o2 CΛ c 00 D11S11B6 CΛ cSRL-5β3 H H C D11S11B7 cSRL-5o4 H CΛ D11SU8B 00 X cSRL-5e5 o D11S11B9 c5RL-5e6 73 C D11S11B4 cSRL-5e10 ro a. D1151190 cSRL-511 Dl 151103 cSRL-512 Dl 151194 cSRL-513 D11S1195 cSRL-514 STS MA OFCllltOMOSOMf* I I STS Name FLpler rango Map ProducI Primer [primor] Annealing Locus Name (II different) bond(s) loch, sire (bp) name Piimar soquonce (nM) lomp.( Q C) D11S1196 cSRL-515 58 Λ D11S1197 cSRL-516 56 00 CΛ H D11S1198 cSRL-5f7 56 H C H D11S1199 cSRI-519 56 m CΛ X D11S1191 cSRL-5111 56 m D11S1192 73 cSRL-5112 56 C D11S1200 cSRL-5g1 56 D11 SI 203 cSRL-5g2 56 D11S1204 cSRL-5g6 56 D11S1205 cSRL-5gB 56 STS MA OPCIIROMOSOMΠ 11 ~ STS Nome FLpter range Map Product Primer [primer] Annealing Locus Name (If dilfβrenl) ba-d(s) tech. size (bp) name Primor soquence nM lem . Q C D11S1206 cSRL-5g9 0.05-0.24 b 335 cSRL-5g9 tA CCCCTTAAATACCACACAATCC 11p15.4-p13 cSRL-5g9-tZ TCACCCCAGTCTTAACCCAC D11S1201 cSRL-5g10 0.24-0.33 b 1GB cSRL-5-olO-tA ACCTCCΛCATCCTCCACΛT CΛ c 11p13-p11.2 cSRL-5g10-l2 CCCATCTAT CTCCACACΛC 00 CΛ D11S1202 cSRL-5g11 0.33-0.50 b 150 cSRL*5g11-IΛ ACAAAAAATCCCAACTACACCA H 11p11.2-q13.1 cSRL-5g11 l2 AACAACTCTCCACCCCTCG H C D11S1209 cSRL-5h2 0.33-0.50 b 260 cSRL-5h2-tΛ TCGATCACCAGCCAACAATA H 11p11.2-q13.1 cSRL-5h2-t2 CCATCCCTCCAACCACAC CΛ 00 X D11S1210 cSRL-5h3 0.85-0.93 b 302 cSRL-5h3-lA AAACαCCCCACrCCTAC m 11 ** .23.3-q24.1 cSRL-5h3 l2 TCTCTTATCGCCCATCGTC m H Dl 1 SI 211 cSRL-5h4 0.60-0.85 b 273 cSRL-5h4-tA CCAAAAΛCCACTACATCATCCA 30 11q14.1-q23.3 cSRL-5h4-l2 CCACCAAATCCATCTTTTΑ C D11S1212 cSRL-5h6 0.60-0.85 b 207 cSRL-5h6-IA TTTACACCCACAAAAACCACTT 11q14.1-q23.3 t cSRL-5h6 l2 AAACTCCAAACTTCCAAACTTC D11S1213 cSRL-5h7 0.05-0.24 b 271 cSRL-5h7-tΛ CCTCTAACACCTCCCTCCTC 11p15.4 p13 cSRL-5h7-l2 CACACTCTCCACCAATCCCA D1IS1214 cSRL-5lι9 0.24-0.33 b 205 cSRL-5h9 IΛ TCAACCAAAACACCCAATCC 11p13-p11.2 cSRL-5h9-l2 ΠTΠGCGCATCTCTCATCA D11S1207 cSRL-5h10 o.eαo.85 I) 328 cSRL-5h10 IΛ ATCCAACCTCCCTCATAAACTT 11q14.1-q23.3 cSRL-5h10-l2 CCAAACCAAAAACCCAAAACT D11S120B cSRL-5h12 0.05-0.24 b 268 cSRL-5h12-IA ACTCΑACCCACCCCAATCT 11p15.4-p13 cSRL-5h12-!2 TACTCCCCTACCTCCATCAC STS MA O CIIUOMOSOMI* I I STS Name FLpler range Map ProducI P nine r [prim tit] Annealing Locus Name (if dillerenl) band(s) lech, size (bp) name Pil er soquence (nM) lβmp.(°C) D11S1216 cSRL-6o2 56 Λ D11S1217 cSRL-6a3 56 σ Λ D11S1218 cSRL-6a4 56 D11S1219 cSRL-6o5 56 Λ oo D11S1220 cSRL-6aG 56 <_ D11S1221 cSRL-6a7 56 73 D11S1215 cSRL-6a12 56 ro D11S1222 cSRL-6b1 52 D11S1225 cSRL-6b2 52 D11S1226 cSRL-6b7 56 ST. MA O CIIROMOSOMΠ 11 c 00 IΛ H H C H 1/1 00 X 30 C STS MA O CHROMOSOMf; 1 I STS Name FLpler range Map ProducI Primer [primer] Annealing Locus Name (il dillerenl) band(s) lech, size (bp) name Primor soquence (nM) lamp (°C) D11S1236 ySL-A13E4 56 D11S1237 ySL-A77g8 54 D11S1238 ySL-B215B10 54 C CD D11S1239 ySL-B34<)9 56 3 ACP2 58 m OO IΛ ACRV1 58 i X ACT 3. 58 73 c ΛHNAK 58 ro AN 2 60 APOA1 56 STS MA OP CHROMOSOMΠ 1 1 STS Name FLpter range Map ProducI Primer [primer] Annealing Locus Name (II dillerenl) ba-d(s) tech. size (bp) name Primer soquence lemp.(°C) 52 BCL1 56 DONF t l C C1 NH 56 OS IΛ H H CΛLCΛ 56 C H CΛLCB 56 IΛ 00 σ * - CΛLCP 58 _x> c CAT 56 CCND1 56 CD20 44 CD3D CD44 56 STS MAP OP CIIUOMQSOMΠ STS Name FLpler range Map Product Primer [primer] Annealing Locus Name (II diflerent) band(s) tech. size (bp) name Primer soquence (nU) lamp.( Q C) CD5 56 CHRM 1 60 Λ CHRM4 50 O Λ CK2Λ 58 CLG 56 oo Λ CLG1 56 3 CNTF CNTF1 56 ι— m COX8 65 t ) CRYAB CRYA2 56 D1 1 S149 60 ST.S MA O CIIROMOSOMR I I m _-_ 50 m rv» STS MA OPCIIKOMOSOMI: 1 I STS Name FLpler ranrje Map ProducI Primer [primer] Annealing Locus Name (II different) band(s) lech, size (bp) name Primer soquence (nM) lamp (°C) D11S860 55 Λ D11S8G1 O Λ D11S862 58 c -i H m D11S863 60 oo VO IΛ X m D11S865 58 m H D11S869E 55 m D11S870 55 en D11SB72 56 D11S873 54 Dl 15874 56 STS MA OP IIROMOSOMΠ I I STS Name FLpler range Map Product Primer (primer) Annealing Locus Name (II different) ba-d(s) lech, site (bp) name Primor soquence (nM) temp.( Q C) D11S875 56 D11S876 56 IΛ c 09 D11S943E 56 IΛ H H D11S944E C 56 m σ IΛ D11S950E X m D11S952E 56 73 C D11S953E 56 rπ rv» a. D11S956 56 DI1S964 56 Ol 1C960 D11S9G9 49 STS MΛI-O CIIROMO.SOMP.11 Λ Λ H H m IΛ X m 73 C STS MA OPCIIROMOSOMΠ 11 . 00 IΛ H H C ro IΛ _r 73 c r- STS MΛ O CIIROMO.SQMP. I I STS Name FLpler range Map Product Primer (primβi] Annealing Locus Name (II dillerenl) band(s) loch, sire (bp) name Piimor soquonco (nM) lemp.(°C) MLP 56 MUC2 50 Λ MYOD1 55 09 Λ H NCAM 66 H C H NFRKB NF-RKB 60 in X m NGK2 56 73 OSBP C 57 IV) PBGD 53 σ. PC 56 PGA3 52 65 STS MA O CIIROMOSOMP. I I O 4 * STS MAP OP CHROMOSOME 1 1 c 09 IΛ H H c H m IΛ _E m rπ H 30 C ι— m r STS MAP or CHROMOSOME 1 1 Map Product Primer (primer) Annealing tech. size (bp) name Primer sequence (nM) lemp.(°C) αbc 507 56 195 50 Ob 178 58 c 00 (/I H a Locations of loci were were determined by F1SII (a), cell hybrid analysis (b). and from the ODD (c). Iliose without regional mapping infoπnation are shown as nd. X m Data Analysis As described above, 371 DNA sequences fragments, determined at one pass accuracy, comprising 116 kb of chromosome 11 derived from cosmid ends were analyzed for the presence of repetitive sequences, simple sequence repeats and similarities to known genes. All of the sequence fragments were subjected to computer analyses for the presence of noteworthy sequence structures (Table 7) . The presence of repetitive sequences was determined using the program FASTA and a repetitive sequence database (Jurka et al., 1992, J. Mol. Evol. 35, 286-291) supplemented with a comprehensive set of di- and tri-nucleotide repeats. A FASTA cutoff score of 100 determined visually was used to recognize repetitive sequences from background random matches. Similarities to known genes were identified with the program BLAST and the GenBank database. Amino acid comparisons were performed by translating DNA sequence fragments into all six potential reading frames and comparing translations to protein sequences in the Swiss-Prot, GenPept or PIR databases using the program BLASTX. Putative exons were identified using the program GRAIL on the Oak Ridge National Laboratory Internet server. The results of these various searches were evaluated numerically and by inspection. The data associated with this project, including DNA sequence file pointers, predicted STS primer sequences, test and mapping results, and in situ hybridization analysis, were stored in a relational database called Genome Notebook specifically designed for this project. DNA sequence and mapping information on other genes was imported into the Genome Notebook database from GDB and GenBank. STS MAP OF CHROMOSOME 1 1 TΛIILF. 7 Analysis of 371 random cosmid end sequences determined by automated fluorescent sequencing Category number percentage subcategory total category Contain repetitive DNA Alu IJNE-1 Middle element repetitive CA repeats others Grail predicted exons Excellent Good Marginal Matches to protein sequences Certain Probable Likely Possible Marginal Note: Repetitive sequence analysis was carried out using FASTA and a customized repetitive sequence database. Similarities to known proteins was determined using BLASTX. Putative exons were deteπnined using GRAIL. Tlie frequency of a sequence fragment containing the element is shown in bold and subtypes of repetitive DNA, potential exons and sequence match quality is shown as well as percentage of total. The last column shows the percentage of sequences within each category The results indicate that the average length of reliable sequence was 312 nucleotides with a standard deviation of 46 nucleotides. Repetitive DNA sequences of some type were found in 150 of these sequences with Alu elements (40%) and LINE-l sequences (27%) being the most frequent. Middle element repetitive sequences (11%) and simple sequence (CA)n repeats (5%) were also detected with reasonable frequency. The neural net based program, GRAIL, was utilized to predict the locations of possible exons and detect putative genes in 34 sequences (9%) ; half of these were rated excellent or good according to reliability estimates used by this program. Analysis for additional possible gene sequences was carried out by computer searches for identity or similarity matches at the nucleotide and amino acid level. Significant matches to known protein sequences were detected for twenty-nine (8%) of the sequence fragments (see, e.g., Table 8). STS MAP OF CHROMOSOME 11 TABLE 8 Results of searching the six-potential coding frames of 371 cosmid end sequences against the protein sequence databases with BLAST-X LD ely significance sequence name map position potential homologue c H H C mitochondrial acetoacetyl-CoA thiolase H UDP-N-acetylglucosaraine-dolichyl-phosphate N- (/I acetylglucosaminephosphotransferase o _T o m m postsynaptic density protein H Zinc finger protein ZFP-37 retrovirus related POL polyprotein opioid binding protein/cell adhesion molecule a. discs large tumor suppressor env polyprotein - feline endogenous virus ECE1 mitochondrial carnitine palraitoyltransferase precursor GATA-3 transcription factor Likely significance sequence name map posmon potential homologue Possible cSRL-2f5-t I lpl5.4-pl3 biglycan C 00 cSRL-3a3-t llpl3-pl l.2 cytochrome P450 IIA6 (Couraarin 7-hydroxylase) cSRL-3a6-t potassium channel protein H C cSRL-4-e2-t phosphomannomutase H m cSRL-4h2-t properdin, mouse i i X cSRL-5bll-t hypothetical 137.7 KD protein in subtelomeric Y' repeat m cSRL-6b4-t Wnt-4 protein H cSRL-7e6-t olfactory receptor protein 50 c Note: Matches are grouped into several categories of likely significance from certain to marginal, a The D 1 ls384 sequence was deteπnined manually and is not part of the 371 sequences determined with automated techniques 0 Two significant matches, from the same gene family, were found to the same cosmid. c This cosmid was derived from hamster based on PCR amplification results. The alignments of nine of the best matches are shown in Figure 11 including matches to mitochondrial acetoacetyl-CoA thiolase, UDP-N-acetylglucosamine- dolichyl-phosphate N-acetylglucosamine phospho- transferase, postsynaptic density protein (and the related discs-large tumor suppressor) , zinc finger protein ZFP-37, retrovirus related pol polyprotein, opioid binding protein/cell adhesion molecule, the env polyprotein from feline endogenous virus ECE1, mitochondrial carnitine palmitoyltransferase II precursor, and the trans-acting T-cell specific transcription factor GATA-3. The zinc finger protein match is probably not of a human genomic DNA origin since PCR amplification with primers predicted from its nucleotide sequence only generate specific products from hamster DNA. One sequence fragment from a cosmid containing the marker D11S384 was determined with manual methods and exactly matched 72 bases of mitochondrial acetoacetyl-CoA thiolase exon six, mapping this gene to llq23.1 or FLpter 0.78. This gene was recently localized independently to chromosome Ilq22.3-q23.1, in agreement with the localization of this sequence, by traditional mapping methods. The detection of a DNA sequence encoding a protein fragment with 97% protein sequence identity (68/70 amino acids) to the hamster UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase strongly suggests that the human homolog of this enzyme, or a highly conserved pseudogene, is located on chromosome llq23.3. The remaining protein sequence matches range from possible to marginally significant. The significance of some of the detected matches is remarkable and makes gene localization to these regions likely. The identification of eight putative new genes from 371 cosmid end sequences corresponds to a 2% gene identification rate and the 19 likely or possible genes corresponds to 5% (Table 8; Figure 11). In comparison, the rate of gene identification from brain cDNA sequencing is about 14%. Strategies for sequencing cDNA libraries suffer from the problem of sequencing the same cDNA multiple times due to the differential abundance of mRNAs. As demonstrated here, random genomic sequencing is associated with a reasonable rate of gene identification (2-5%) coupled with direct gene mapping, and thus is considered to be an advantageous strategy for further characterization of cosmid and YAC clone maps. STSs prepared by the methods described herein, will provide a useful reagent set for further physical mapping, for constructing YAC contigs using STS content mapping, and for further DNA sequence analysis. In addition, automated fluorescent sequencing of randomly chosen cosmid clones is a rapid and powerful tool for generating PCR detectable markers as well as defining the locations of putative genes. Theoretical analysis of the strategy of STS content mapping (Arratia et al., 1991, Genomics. 11:806-827; Palazzolo et al., 1991, Proc. Natl. Acad. Sci.. USA. 88:8034-8038) suggests that this number of unique and uniformly distributed markers will serve as an appropriate starting point for future physical analysis. In summary, sequences were determined from the ends of chromosome 11 specific cosmids by automated sequencing without intermediate subcloning. The STSs and cosmids were mapped by in situ hybridization, somatic cell hybrid analysis or both. This effort generated 370 STSs specific for human chromosome 11 and regionally mapped most of them. Sixty-eight percent of these STSs (251/370) were produced from new chromosome 11 sequences; 18% (68/370) represented sequences derived from cloned genes; 8% (29/370) were based upon STS markers deposited and available from GDB. The latter were retested with our set of standard conditions to allow integration of this map with the results of other groups. While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed. Sequence Listing SEQ ID NO:l GGTCAAGCTC AGCAACATGA SEQ ID NO:2 TGCTTTGTGA CCATCGAGAG SEQ ID NO:3 CAGCAGATGG TCAAGCAAAA SEQ ID NO:4 ACTCCTGACA CCACCACCTC SEQ ID NO:5 TCGCTCACTG ACTCGCTG SEQ ID NO:6 AGCCCTCCCG TATCGTAGTT SEQ ID NO:7 CTTGAGAGCC TTCAACCCAG SEQ ID NO:8 AACTGGGCGG AGTTAGGG

Claims: That which is claimed is: 1. A method for sequencing complex genomes, said method comprising: (1) sequencing the end-specific nucleotides of each member of a library of cosmid clones, wherein said cosmid clones are prepared by inserting genomic DNA fragments into cosmid vectors, and wherein the cosmid vectors include sequences of nucleotides that flank at least one end of the inserted DNA, and that serve as transcription initiation sites for the synthesis of nucleic acids specific to the ends of the inserted DNA, and (2) assembling a sequence sampled map by correlating the end-specific nucleotide sequence information with the relative spatial relationship between the cosmids. 2. A method according to Claim 1 wherein the relative spatial relationship between the cosmids has been determined prior to said sequencing the end-specific nucleotides of each member of a library of cosmid clones. 3. A method according to Claim 1 wherein the relative spatial relationship between the cosmids is determined by the cosmid multiplex analysis method. 4. A method according to Claim 1 wherein the relative spatial relationship between the cosmids is determined by restriction-fragment-length mapping of the cosmids. 5. A method according to Claim 1 wherein at least 100 base pairs of end-specific nucleotide sequences are determined. 6. A method according to Claim 1 wherein said cosmid clones are generated in cosmid vectors allowing for the synthesis of end-specific nucleic acid sequences directly from at least one end of DNA fragments inserted therein. 7. A method according to Claim 6 wherein said cosmid vectors comprise at least one promoter specific for a bacteriophage RNA polymerase and a cloning site allowing for the insertion of DNA fragments, said promoter being positioned operatively for transcription of a DNA fragment inserted into said cloning site. 8. A method according to Claim 7 wherein said cosmid vectors comprise two oppositely oriented promoters, each of which is specific for a bacteriophage RNA polymerase, positioned on two sides of said cloning site, operatively for transcription of a DNA fragment inserted into said cloning site. 9. A method according to Claim 8 wherein each of said bacteriophage RNA polymerase-specific promoters is selected from the group consisting of promoters specific for bacteriophage T7 RNA polymerase, and promoters specific for bacteriophage T3 RNA polymerase. 10. A method according to Claim 9 wherein said cosmid vector is selected from the group consisting of pWE8, pWElO, pWE15, and pWE16. 11. A method according to Claim 6 wherein said cosmid vectors comprise at least two cos sites. 12. A method according to Claim 11 wherein said cos sites are separated by unique restriction sites. 13. A method according to Claim 12 wherein said cosmid vector is selected from the group consisting of sCOS-1, sCOS-2, sCOS-4, and derivatives thereof. 14. A method for sequencing complex genomes, said method comprising: (1) preparing a genomic library of cosmid clones by inserting DNA fragments from said genome into cosmid vectors, wherein the cosmid vectors include sequences of nucleotides that flank at least one end of the inserted DNA, and that serve as transcription initiation sites for the synthesis of end-specific probes, (2) arranging the cosmid clones, whereby each clone may be identified and replicas of said arrangement may be reproduced, (3) pooling portions of said cosmid clones and synthesizing pools of mixed end-specific probes from the DNA inserts that have been prepared from said pooled clones, wherein each pool contains fewer than all of the cosmid clones in the library, but all of the cosmid clones in the library are included in at least one pool, (4) hybridizing each pool of probes to a replica of said arranged cosmid clones and identifying the cosmid clones in each replica that hybridize to the probes, wherein said identified clones include the pooled cosmid clones and cosmid clones that contain DNA inserts that overlap with the DNA inserts in the pooled clones, (5) identifying the cosmid clones from among those identified in step (4) that hybridize to two or more pools of probes, thereby identifying groups of cosmid clones that include overlapping DNA, (6) assembling contigs from said groups, and (7) sequencing the fragment ends of the DNA inserts of each of the overlapping cosmid clones. 15. A method according to Claim 14 wherein cross-hybridizing clones are identified by comparing the data sets obtained from two groups of cosmid clones containing at least one common clone, and repeating the pairwise comparison with other groups of clones containing at least one common clone. 16. A method according to Claim 14 wherein said cosmid clones are pooled according to the rows and columns of a two-dimensional matrix, and said mixed end-specific RNA sequences are hybridized to a replica of the entire matrix. 17. A method according to Claim 14 wherein said cosmid clones are pooled according to the planes intersecting with a three-dimensional matrix, and said mixed end-specific RNA sequences are hybridized to a replica of the entire matrix. 18. A method according to Claim 14 wherein said cosmid clones are generated in cosmid vectors allowing for the synthesis of end-specific RNA sequences directly from at least one end of DNA fragments inserted therein. 19. A method according to Claim 18 wherein said cosmid vectors comprise at least one promoter specific for a bacteriophage RNA polymerase and a cloning site allowing for the insertion of DNA fragments, said promoter being positioned operatively for transcription of a DNA fragment inserted into said cloning site. 20. A method according to Claim 14 wherein said cosmid vectors comprise two oppositely oriented promoters, each of which is specific for a bacteriophage RNA polymerase, positioned on two sides of said cloning site, operatively for transcription of a DNA fragment inserted into said cloning site.

Inventors: EVANS GLEN A, SMITH MICHAEL W, EVANS, GLEN, A., SMITH, MICHAEL, W.
Applicants: SALK INST FOR BIOLOGICAL STUDI, THE SALK INSTITUTE FOR BIOLOGICAL STUDIES
Application year: 1994
Main classification: C12Q1
IPC codes: C07K14, C12N15, C12Q1